System and method for controlling thermal cycler modules

ABSTRACT

Systems and methods for processing and analyzing samples are disclosed. The system may process samples, such as biological fluids, using assay cartridges which can be processed at different processing locations. In some cases, the system can be used for PCR processing. The different processing locations may include a preparation location where samples can be prepared and an analysis location where samples can be analyzed. To assist with the preparation of samples, the system may also include a number of processing stations which may include processing lanes. During the analysis of samples, in some cases, thermal cycler modules and an appropriate optical detection system can be used to detect the presence or absence of certain nucleic acid sequences in the samples. The system can be used to accurately and rapidly process samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/747,952, filed on Jan. 23, 2013, which is a continuation of PCTApplication No. PCT/US 2011/045107 filed Jul. 22, 2011, which claimspriority to U.S. Provisional Application No. 61/367,343, filed on Jul.23, 2010, the disclosures of which are hereby incorporated by referencein their entirety for all purposes.

BACKGROUND

Many nucleic acid sequences have clinical relevance. For example,nucleic acid sequences associated with infectious organisms provideindications of the presence of an infection by the organism. Nucleicacid sequences not normally expressed in a patient sample may indicateactivation of pathways associated with a disease or other conditions.Still other nucleic acid sequences may indicate differences in apatient's likely response to proposed therapies.

Determination of clinically relevant nucleic acids generally depends oncontrolled amplification of specific nucleic acid sequences anddetection of the amplification products. Amplification improvesanalytical sensitivity by generating sufficient copies of nucleic acidsfound in the sample for ready determination. Amplification may alsoimprove analytical specificity by selectively generating only thosenucleic acids of clinical interest. A problem with amplification-baseddeterminations, particularly when amplification generates large numbersof copies of a target nucleic acid sequence, is the possibility thatsome of these copies from one sample might contaminate other samples toproduce apparently elevated results where none of the target nucleicacid sequence was originally present in the sample.

Other sources of contamination could affect nucleic acid determinations.Carryover between samples can contribute contaminating material. Anamplification mixture may receive contaminating materials fromenvironmental sources transferred on surfaces or by laboratorytechnicians or by aerosols. In some cases, unintended transfers ofreagents, such as inappropriate amplification primers, may contaminatemixtures and cause erroneous results. Amplification mixtures may alsoretain interfering substances originally present in the sample throughincomplete purification of target nucleic acids. Thus, there is a needfor automation of nucleic acid analysis that avoids transfer andretention of contaminating material from a variety of sources.

Clinical laboratory workflow is a consequence of medical care deliveryand varies between institutions. A clinic or large group practice maygenerate patient specimens throughout the course of a day at arelatively constant rate. In contrast, a clinical reference laboratorymay receive all of its specimens in one or two deliveries and a largehospital may generate specimens through a large blood draw in themorning supplemented by an irregular stream of samples throughout theday. Most nucleic acid analysis specimens arrive at a clinicallaboratory in a sequence unrelated to the type of requested assay. Insome cases, selected specimens may be of high priority with immediate orcritical treatment decisions dependent on the outcome. Other specimensmay be of more routine priority. Non-specimen samples such as laboratorycontrols may be interspersed among the clinical specimens according toindividual laboratory practice. In some cases, exhaustion of reagents orof particular lots of reagents may dictate the insertion of controls andcalibration samples irrespective of other samples in queue.

Thus, there is a need for an analytical system having flexible andadjustable operating capabilities to match the unpredictable demand ofclinical laboratories.

Nucleic acid analysis determines multiple analytes from diverse sourceorganisms using a mix of specimen types. These inputs drive diverseprocessing requirements. For example, RNA and DNA have differentchemical properties and stabilities; their preparation may use differentprocessing regimens, different enzymes, and different thermalconditions. Both the base sequence and the length of target analytesaffect binding energy, and hence processing. The length and sequence ofcomplementary oligonucleotides used for amplification further affectamplification conditions.

Different source organisms for analytical targets may require differentsteps to release or isolate the nucleic acid sequences. For example,release of DNA sequences from gram positive bacteria might use elevatedtemperatures not used for release of DNA sequences from relativelylabile white blood cells.

Thus, there is a need for an analytical system able to freely intermix avariety of processing protocols, each composed of a variety ofprocessing steps. Technologies exist that attempt to address some of theissues described above.

Russel/Higuchi in U.S. Pat. No. 5,994,056, Homogeneous Methods forNucleic Acid Amplification and Detection, described improved methods fornucleic acid detection using methods such as the polymerase chainreaction (PCR). Higuchi described methods for simultaneous amplificationand detection to enhance the speed and accuracy of prior methods. Themethods provide means for monitoring the increase in product DNA duringan amplification reaction. According to the description, amplifiednucleic acids are detected without opening the reaction vessel once theamplification reaction is initiated and without any additional handlingor manipulative steps subsequent to the reaction.

K. Rudi et al. described a Rapid, Universal Method to Isolate PCR-ReadyDNA Using Magnetic Beads in BioTechniques 22(3) 506-511, March 1997.Rudi et al. described application of a magnetic bead-based kit for rapidDNA isolation (Dynabeads® DNA DIRECT™; Dynal A. S.) to diverse organismsand tissues to produce a general approach for the purification ofPCR-ready DNA. DNA suitable for PCR was prepared in less than 30minutes.

Systems that automate nucleic acid analysis have a long history.Integrated platforms demonstrated the entire range of automatedanalytical and preparative steps, including isolation of nucleic acid,amplification of the isolated material, and detection of theamplification products.

For example, Bienhaus et al. in U.S. Pat. No. 5,746,978, Device forTreating Nucleic Acids from a Sample, described a single device to linktreatment steps that separate nucleic acids from other sample componentswith steps for amplification of the nucleic acids. The device includedreaction chambers for individual treatment steps with the outlet of onechamber attached to inlet of another. A conventional pipettinginstrument transferred both the nucleic acid-containing sample liquidand all possibly necessary reagents from sample and reagent storagecontainers into the device. Bienhaus et al. described magneticseparation, amplification by PCR or NASBA, and using a hybridizationprobe complementary to the PCR amplificate in a detection reactionmeasured using an ES analyzer (manufactured by Boehringer Mannheim).

P. Belgrader, et al. described Automated DNA Purification andAmplification from Blood-Stained Cards Using a Robotic Workstation inBioTechniques 19(3) 427-432 1995. Belgrader et al. introduced aprototype which could perform coupled DNA purification and amplificationthat required no user participation once the process was initiated. Themethod was implemented into a high throughput automated system using aBiomek® 1000 robotic workstation (Beckman Instruments) using phenol andisopropanol to purify DNA on blood-stained cards. The Biomek® 1000performed DNA purification and amplification using an HCU (Biomek®on-board heater-cooler unit) as a thermal cycler. Belgrader et al.described that the next objective was to integrate a detection step fora completely automated DNA typing system.

Patrick Merel et al. described Completely Automated Extraction of DNAfrom Whole Blood in Clinical Chemistry 42, No. 8, p 1285-6 1996. Merelet al. disclosed using the Biomek® 2000 (Beckman Instruments) and DNADIRECT™ (Dynal France S.A.) in combination to fully automate the DNAextraction procedure using magnetic particle separation. Merel et al.used several different PCR protocols to evaluate the quantity andquality of the DNA obtained. Merel et al. routinely used the describedmaterials for a 10-min automated DNA extraction procedure, a 10-minautomated PCR setup step for 96 tubes, PCR for 80 min, and a simpleelectrophoresis analysis of 15 min.

Ammann et al. U.S. Pat. No. 6,335,166 Automated Process for Isolatingand Amplifying a Target Nucleic Acid Sequence described an automatedanalyzer including multiple stations, or modules, in which discreteaspects of the assay are performed on fluid samples contained inreaction receptacles. The analyzer includes stations for automaticallypreparing a specimen sample, incubating the sample at prescribedtemperatures for prescribed periods, preforming an analyte isolationprocedure, and ascertaining the presence of a target analyte. Anautomated receptacle transporting system moves the reaction receptaclesfrom one station to the next. Ammann also describes a method forperforming an automated diagnostic assay includes an automated processfor isolating and amplifying a target analyte. The process is performedby automatically moving each of a plurality of reaction receptaclescontaining a solid support material and a fluid sample between stationsfor incubating the contents of the reaction receptacle and forseparating the target analyte bound to the solid support from the fluidsample. An amplification reagent is added to the separated analyte afterthe analyte separation step and before a final incubation step.

Even though such automated systems have been available, furtherimprovements are desirable. In particular, multiple sources ofcontamination continues to risk erroneous results. Further, complexitiesof multi-step processes needed for complete nucleic acid analysis canproduce processing bottlenecks and degrade repeatability, limitinganswer reporting turnaround and processing flexibility. Limited answerreporting turnaround may increase the time to institute proper clinicaltreatment. Lack of processing flexibility limits support for variationsin assay protocols for a broad and expandable test menu. Lack ofprocessing flexibility may also force laboratories to sequence or batchsamples and reagents in a manner at odds with clinical need.

Embodiments of the invention address these and other problems,individually and collectively.

SUMMARY

Embodiments of the invention are directed to systems, methods, anddevices associated with the processing of samples, which may contain DNAor RNA. Embodiments of the invention include a fully-automated, randomaccess system for determining specific nucleic acid sequences.

One embodiment of the invention is directed to a system for processing asample. The system comprises a preparation location suitable forprocessing the sample in an assay cartridge including a firstcompartment and a second compartment. The system also includes a firstpipettor configured to transfer liquids from the first compartment tothe second compartment of the assay cartridge. The system furthercomprises a materials storage location that is distinct from thepreparation location. It also comprises a second pipettor disposed totravel between the materials storage location and the preparationlocation. The system also comprises a controller configured to directthe first pipettor to transfer a first reagent from the firstcompartment to the second compartment of the assay cartridge, and todirect the second pipettor to transfer a second reagent from thematerials storage location to the second compartment.

Another embodiment of the invention is directed to a method comprisingproviding an assay cartridge comprising a first compartment and a secondcompartment with a cartridge guide, transferring a first reagent from afirst compartment to a second compartment in an assay cartridge using afirst pipettor at a preparation location, and transferring a secondreagent from a reagent pack in a reagent storage unit to the secondcompartment using a second pipettor.

Another embodiment of the invention is directed to a sensor systemcomprising a mandrel and a sensing circuit. The sensing circuit isconfigured to determine a characteristic of the mandrel or of anextension element on the mandrel. The sensing circuit comprises one ormore sensor channels, coupled to a processor configured to determine thecharacteristic of the extension element based on the error signal.

Another embodiment of the invention is directed to a system forprocessing a sample. The system comprises a first pipettor, a secondpipettor, and a controller operatively coupled to the first pipettor andto the second pipettor. The controller is configured to direct the firstpipettor to transfer a fluid from a first compartment in an assaycartridge or from a reagent pack in a reagent storage unit to a reactionvessel in the assay cartridge, and to direct the second pipettor toremove the reaction vessel from the assay cartridge.

Another embodiment of the invention is directed to a method comprising:providing an assay cartridge comprising a first compartment and a secondcompartment with a cartridge guide, transferring a first reagent from afirst compartment or from a reagent pack in a reagent storage unit to areaction vessel in an assay cartridge using a first pipettor, andremoving the reaction vessel from the assay cartridge using the secondpipettor.

Another embodiment of the invention is directed to a sensor systemconfigured to determine at least two properties associated with amandrel. The sensor system comprises a sensing circuit comprising aprocessor and a mandrel. The sensing circuit is configured to generate afirst signal and a second signal, each of the first signal and thesecond signal relating to at least one of resistance, capacitance, andinductance of the mandrel. The processor is further configured tocompare the first signal to a first stored reference value to determinecontact of an extension element with a liquid. The processor is furtherconfigured to compare the second signal to a second stored referencevalue to determine one of the presence of an extension element on themandrel, the fill level of the extension element, or the proximity ofthe mandrel to a conductive target.

Another embodiment of the invention is directed to a system, which canbe for determining the presence of a nucleic acid in a sample. Thesystem may comprise a cartridge loading unit to accept a plurality ofassay cartridges. The cartridge loading unit can include a storagelocation to support the plurality of assay cartridges, a loading lanecoupled to the storage location, and a loading transport coupled to thestorage location and to the loading lane and configured to move an assaycartridge from the storage location to the loading lane. The system canalso include a plurality of processing lanes to process an assaycartridge, each processing lane configured to operate on an assaycartridge, a shuttle to move the assay cartridge among the loading laneand the plurality of processing lanes. The shuttle can be positionablein alignment with the loading lane and in alignment with each of theplurality of processing lanes. A controller can be operatively coupledto the loading transport, to the shuttle, and to the plurality ofprocessing lanes.

Another embodiment of the invention is directed to a method comprisingloading a plurality of assay cartridges into a storage location in acartridge loading unit, wherein each assay cartridge includes a reactionwell and a reagent well containing a reagent. The method also includesmoving an assay cartridge of the plurality of assay cartridges to aloading lane using a loading transport, moving the assay cartridge to ashuttle, and moving the assay cartridge to one of a plurality ofprocessing lanes. Each processing lane can be configured to process theassay cartridge using a different process.

Another embodiment of the invention is directed to a system comprising afirst processing lane, a second processing lane, a third processinglane, and a transfer shuttle operatively coupled to the first, second,and third processing lanes. The system further comprises a controlleroperatively coupled to each of the first, second and third processinglanes and the transfer shuttle. The controller can be configured toexecute a first protocol and a second protocol. The controller inexecuting the first protocol directs the transfer shuttle to move afirst assay cartridge from the first processing lane to the secondprocessing lane. The controller in executing the second protocol directsthe transfer shuttle to move a second assay cartridge from the firstprocessing lane to the third processing lane without moving the assaycartridge to the second processing lane.

Another embodiment of the invention is directed to a method comprising:executing a first protocol by a controller, wherein in the firstprotocol, the controller directs a transfer shuttle to move a firstassay cartridge from the first processing lane to the second processinglane; and executing a second protocol by the controller, wherein in thesecond protocol, the controller directs the transfer shuttle to move asecond assay cartridge from the first processing lane to the thirdprocessing lane without moving the assay cartridge to the secondprocessing lane.

Another embodiment of the invention is directed to a system comprising apreparation location for processing samples, a reaction vessel forcontaining the processed sample, an analysis location for characterizingthe processed sample, and a transport device for transferring thereaction vessel between the preparation location and the analysislocation. The system may also comprise a plurality of non-identicalprocessing lanes in the preparation location, the processing lanesconfigured to perform different processing functions, and a plurality ofidentical analytical units in the analysis location.

Another embodiment of the invention is directed to a method comprisingloading a sample into a system, and loading an assay cartridge into apreparation location. The assay cartridge includes a reaction well and acompartment. A reaction vessel is in the compartment. The method alsoincludes extracting the nucleic acid in the reaction well, transferringthe extracted nucleic acid from the reaction well to the reactionvessel, moving the reaction vessel to a thermal cycler module, anddetecting the nucleic acid in the thermal cycler module.

Another embodiment of the invention is directed to a system fordetermining the presence of a nucleic acid in a sample, the systemcomprising a first processing lane configured to perform operations on asample in an assay cartridge, a transfer shuttle configured to moveassay cartridges into and out of the first processing lane, and acontroller to direct operation of the system. The controller can beoperatively coupled to the first processing lane and to the transfershuttle, and can be configured to execute a first protocol and a secondprotocol. The controller, in executing the first protocol, directs thetransfer shuttle to move a first assay cartridge into the firstprocessing lane, and after a fixed interval, directs the transfershuttle to move the first assay cartridge out of the first processinglane, and within the fixed interval directs the first processing lane toexecute a first sequence of operations. The controller, in executing thesecond protocol, directs the transfer shuttle to move a second assaycartridge into the first processing lane, after the fixed interval,directs the transfer shuttle to move the second assay cartridge out ofthe first processing lane, and directs the first processing lane toexecute a second sequence of operations that differs from the firstsequence of operations.

Another embodiment of the invention is directed to a method comprisingexecuting a first protocol by a controller, to direct a transfer shuttleto move a first assay cartridge into a first processing lane, after afixed interval, direct the transfer shuttle to move the first assaycartridge out of the first processing lane, and within the fixedinterval direct the first processing lane to execute a first sequence ofoperations. The method also includes executing a second protocol by thecontroller, to direct the transfer shuttle to move a second assaycartridge into the first processing lane, after the fixed interval,direct the transfer shuttle to move the second assay cartridge out ofthe first processing lane, and direct the first processing lane toexecute a second sequence of operations that differs from the firstsequence of operations.

Another embodiment of the invention can be directed to a pipettor fortransferring liquids on an automated instrument, comprising a linearactuator and a piston enclosed within a barrel. The piston comprises afluid tight seal with the inner wall of the barrel, the piston and thebarrel cooperatively configured to allow movement of the piston withinthe barrel. The pipettor can comprise a compliant coupling interposedbetween the linear actuator and the piston, the compliant couplinghaving a first connecting feature affixing the compliant coupling to thelinear actuator, a second connecting feature affixing the compliantcoupling to the piston, and a compressible member interposed between thefirst connecting feature and the second connecting feature.

Another embodiment of the invention is directed to an assay cartridgecomprising a reaction well including a first sidewall, a secondsidewall, a first endwall, a second endwall, and a well floor arrangedto receive a reaction mixture. The first sidewall, the second sidewall,the first endwall and the second endwall form an open end. The firstendwall includes a first segment and a second segment. The first andsecond segment are joined by a bend, and at least one of the firstsegment and second segment is tapered so that the cross section of thereaction well decreases closer to the well floor.

Another embodiment of the invention is directed to a method for mixingthe contents of a well. The method comprises directing a pipettor to afirst location in an assay cartridge having a well with an endwallcomprising a segment, a first sidewall, and a second sidewall, where thesegment of the endwall extends towards the center of the well at anangle relative to the vertical axis and has a radius about a mid-planeto create a culvert, the mid-plane being defined by the first sidewalland the second sidewall. The method also includes dispensing a liquidfrom the pipettor onto the culvert of the well, wherein the radius ofthe culvert collects the dispensed liquid and directs the dispensedliquid towards the midline of the culvert such that turbulence isinduced in the flow of the dispensed liquid.

Another embodiment of the invention is directed to a cartridge loadingunit for loading assay cartridges onto an automated system. It includesa presentation lane including a carriage to receive an assay cartridge,the presentation lane configured to transport the assay cartridge intothe automated system for processing. It also includes a first loadinglane including a cavity to receive a DNA assay cartridge and transferthe DNA assay cartridge to the carriage of the presentation lane. TheDNA assay cartridge includes a reaction well and a reagent compartment,the reagent compartment containing a reagent used for DNA extractionfrom a sample.

Another embodiment of the invention can be directed to an automatedanalyzer comprising a pipettor, a reagent pack comprising a wellcontaining a reagent, and a reagent storage unit. The reagent storageunit is configured to hold the reagent pack, and includes a cavitycontaining the reagent pack, a latch arranged about the cavity, thelatch configured to secure and align the reagent pack within the cavity,the latch including a releasing feature, a cover disposed over thecavity and latch, the cover including a first aperture and a secondaperture, wherein the first aperture aligns over the well of the reagentpack thereby providing the pipettor access to the reagent contained inthe well. The second aperture aligns over the releasing feature therebyproviding the pipettor access to actuate the releasing feature tounsecure the reagent pack from the latch.

Another embodiment of the invention is directed to a method comprisingaligning a consumable pack in a storage unit. The consumable packcomprises consumables that are manipulated using a pipettor. The methodalso includes securing the consumable pack within the storage unit byengaging a latch having a releasing feature with a mating feature of theconsumable pack, and releasing the consumable pack by aligning thepipettor with the releasing feature, moving the pipettor towards thereleasing feature, and contacting the releasing feature with thepipettor. This causes the latch to disengage from the mating of theconsumable pack.

Another embodiment of the invention is directed to a reagent cartridgecomprising a containment section that comprises a horizontally planarcontainment floor and a containment wall that extends vertically fromthe periphery of the containment floor, the floor including an accessopening of a reagent receptacle. It also includes a gripping handle thatis attached to an isolation portion, the isolation portion attached tothe containment section and thereby providing a separation betweengripping handle and the reagent receptacle, and a memory unit.

Another embodiment of the invention is directed to a system comprising:a movable cartridge carriage configured to engage an assay cartridge.The assay cartridge comprises a well containing a magneticallyresponsive particle, the well including a wall at an angle relative tothe vertical axis. The system also includes a movable magnet trolley,the movable magnet trolley comprising a separation magnet mounted at anangle complementary to the assay cartridge wall angle, and a reversiblecoupling device configured to reversibly join the movable cartridgecarriage and the movable magnet trolley. The separation magnet isaligned in proximity to the assay cartridge wall when the movablecartridge carriage is coupled to the movable magnet trolley.

Another embodiment of the invention is directed to an assay cartridgeincluding a reaction well, a pipette tip, and a reagent well in a lineararrangement, wherein the pipette tip lies between the reaction well andthe reagent well; and a processing lane comprising a lane heater,wherein the lane heater comprises a plurality of heating zones that arein thermal communication with the assay cartridge. The first heatingzone is juxtaposed with the reaction well and a second heating zone isjuxtaposed with the reagent well.

Another embodiment of the invention is directed to a system forprocessing an assay cartridge. The system comprises a first assaycartridge comprising reagents for processing a first analyte, a secondassay cartridge comprising reagents for processing a second analyte, afirst processing lane comprising a heating assembly configured totransfer heat to an assay cartridge raise the temperature of an assaycartridge, and a second processing lane comprising a heating assemblyconfigured to transfer heat to an assay cartridge to maintain thetemperature of an assay cartridge. The temperature of the first assaycartridge may be raised to a first temperature in the first processinglane and the first temperature maintained in the second processing laneand the second assay cartridge may be raised to a second temperature inthe first processing lane and the second temperature maintained in thesecond processing lane. The first and second temperatures are different.

Another embodiment of the invention can be directed to an assaycartridge comprising an elongated body comprising a distal end and aproximal end, and a plurality of compartments arranged linearly betweenthe distal end and the proximal end. At least one of the compartments isa reaction well. The reaction well comprises first and second sidewalls,and first and second endwalls, and a well floor joining at least thefirst and second endwalls. The first endwall comprises a plurality ofbends.

Another embodiment of the invention is directed to a cartridge loadingunit for loading assay cartridges onto an automated system. Thecartridge loading unit comprises a rail for supporting an assaycartridge. The assay cartridge comprises a keying feature. The cartridgeloading unit also comprises an identification bar, and a baseplatecoupled to the rail and identification bar. The identification bar ispositioned on the baseplate to mate with the keying structure, therebypermitting the assay cartridge to rest on the rail.

Another embodiment of the invention is directed to a method comprising:placing an assay cartridge in the cartridge loading unit, and mating akeying feature of the assay cartridge with the identification bar. Themating of the keying feature of the assay cartridge with theconfiguration bar allows the assay cartridge to rest on a rail in thecartridge loading unit in alignment with a pusher. The method alsoincludes propelling the aligned cartridge towards a presentation laneusing the pusher.

Another embodiment of the invention is directed to a method comprising:aligning a probe with an aperture in a storage unit that holdsconsumable packs, inserting the probe through the aperture, and pushinga latch as the probe is inserted through the aperture. This causes thelatch to disengage from a latch pocket of a consumable pack held withinthe storage unit.

Another embodiment of the invention is directed to a system comprising:a slidable cartridge carriage configured to engage an assay cartridge,the cartridge carriage engaging a carriage track; a slidable magnettrolley, the slidable magnet trolley engaging the carriage track andcomprising a separation magnet; and a reversible coupling deviceconfigured to reversibly couple slidable cartridge carriage and theslidable magnet trolley.

Another embodiment of the invention is directed to a system comprising:an assay cartridge comprising a plurality of compartments; and a laneheater, wherein the lane heater is cooperatively configured with theassay cartridge. The lane heater is in thermal contact with a pluralityof the compartments of the assay cartridge when the assay cartridge isengaged with the lane heater.

Another embodiment of the invention is directed to a system comprising alinear track, a pipetting arm coupled to the linear track, and a slidelock manipulator coupled to the linear track and configured to extendaway from the linear track and retract towards the linear track.

Another embodiment of the invention is directed to a method comprising:acquiring a reaction vessel with a pipetting arm, opening an analyticalunit with a slide lock manipulator, aligning the pipetting arm with theanalytical unit; and releasing the reaction vessel from the pipettingarm.

Another embodiment of the invention is directed to a thermal cyclermodule for performing real time PCR within a PCR reaction vessel. It maycomprise a thermal block comprising a receptacle for receiving a PCRreaction vessel, and a slidable lid. The lid overlaps with the thermalblock and has an open position and a closed position. It is capable ofmoving between the open and closed positions. It can also include anexcitation optics assembly, the excitation optics assembly configured topass excitation light to the PCR reaction vessel when the PCR reactionvessel is located in the receptacle, and an emission optics assembly,the emission optics assembly is configured to receive light from the PCRreaction vessel when the PCR reaction vessel is located in thereceptacle in the thermal block.

Another embodiment of the invention is directed to a plurality ofthermal cycler modules. Each thermal cycler module includes a thermalblock having a top surface and a defined receptacle. The receptacle canbe tapered to conform to a reaction vessel. Each thermal cycler modulealso comprises a heater thermally coupled to the thermal block, atemperature sensor thermally coupled to the thermal block, and atemperature controller electrically coupled to the heater and to thetemperature sensor and configured to cycle the thermal block between atleast two temperatures independently of the other thermal cyclermodules. Each thermal cycler module also includes an excitation opticsassembly. The excitation optics assembly is configured to passexcitation light to the reaction vessel when the reaction vessel islocated in the receptacle in the thermal block. Each thermal cyclermodule may also include an emission optics assembly, wherein theemission optics assembly is configured to receive light from thereaction vessel when the reaction vessel is located in the receptacle inthe thermal block.

Another embodiment of the invention is directed to a method forconducting a PCR reaction process using a thermal cycler module, thethermal cycler module comprising a thermal block, and the thermal blockcomprising a receptacle configured to receive a PCR reaction vessel, anda slidable lid. The method comprises: inserting the PCR reaction vesselin the receptacle; and sliding the slidable lid from the open positionto the closed position.

Another embodiment of the invention is directed to a vessel for realtime PCR comprising: a radially symmetrical reaction base, and a plugcomprising a handling feature, the handling feature configured toreceive a pipette mandrel, wherein the reaction base comprises an uppercylindrical portion that receives the plug and a lower portion, andwherein the lower portion opens into the upper cylindrical portion andcomprises a frustum of a conical shape.

Another embodiment of the invention is directed to a system comprising:a plurality of thermal cycler modules, each thermal cycler moduleincluding a thermal block having a top surface and a receptacle, thereceptacle tapered to conform to a reaction vessel; a heater thermallycoupled to the thermal block; a temperature sensor thermally coupled tothe thermal block; and, a temperature controller electrically coupled tothe heater and to the temperature sensor and configured to cycle thethermal block between at least two temperatures independently of otherthermal blocks in other thermal cycler units; an excitation opticsassembly, the excitation optics assembly configured to pass excitationlight to the reaction vessel when the reaction vessel is located in thereceptacle in the thermal block; and an emission optics assembly, theemission optics assembly configured to receive light from the reactionvessel when the reaction vessel is located in the receptacle in thethermal block.

Another embodiment of the invention can be directed to a process fordetermining a nucleic acid in a sample using a system including aprocessing area and a thermal cycler, the process comprising the stepsof: providing in the processing area a vessel plug with a grippingfeature and a vessel base configured to lockably engage with the vesselplug; pipetting an amplification reagent to the vessel base with apipette tip held on a mandrel; pipetting the nucleic acid to the vesselbase; lifting the vessel plug using the mandrel to grip the grippingfeature; engaging the vessel plug to the vessel base; and moving theengaged vessel plug and vessel base to the thermal cycler.

Another embodiment of the invention is directed to vessel for real timePCR comprising: a radially symmetrical reaction base, and a plugcomprising a handling feature, the handling feature configured toreceive a pipette mandrel.

Another embodiment of the invention is directed to a method foroperating a thermal cycler module, the method comprising: obtaining apredetermined temperature vs. time profile associated with a selectedthermal cycler module in an array of thermal cycler modules, the arrayof thermal cycler modules comprising the selected thermal cycler moduleand a set of thermal cycler modules; and controlling, by a processor,the thermal cycler modules in the set of thermal cycler modules so thattheir performance matches the predetermined temperature vs. timeprofile, each of the thermal cycler modules in the set of thermal cyclermodules being controlled using a source of variation between the thermalcycler modules in the array.

Another embodiment of the invention is directed to a method of driving afirst thermal cycler in a predetermined thermal profile (B(t)), thefirst thermal cycler including a thermal block, a heater thermallycoupled to the thermal block, and a blower to direct air to the thermalblock, the method comprising: determining the rate of change of thethermal block temperature with respect to time (dB/dt) as a function ofheater output (h_(a)), of blower heat transfer (k), and of ambienttemperature (Ta); measuring the thermal block temperature; measuring theambient temperature at the thermal cycler; and adjusting one of theheater output and the blower heat transfer according to a modeledrelationship of:

dB/dt=h _(a) +k(Ta−B(t)).

These and other embodiments of the invention are described in furtherdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a) shows a front perspective view of an instrument according toan embodiment of the invention.

FIG. 1( b) shows a top plan view of the layout of the components of theinstrument.

FIG. 1( c) is a top plan view of the instrument.

FIG. 1( d) shows a partial front view of the instrument.

FIG. 2( a) shows a front perspective view of a sample presentation unitaccording to an embodiment of the invention.

FIG. 2( b) shows a front perspective view of a sample presentation unitpusher cartridge according to an embodiment of the invention.

FIG. 3( a) shows a front, perspective view of a sample pipettoraccording to an embodiment of the invention.

FIG. 3( b) shows a front perspective view of the sample pipettor in moredetail.

FIG. 3( c) shows a perspective view of a compliant coupling.

FIG. 4( a)-1 shows a top perspective view of an assay cartridgeaccording to one embodiment of the invention.

FIG. 4( a)-2 shows a top perspective view of an assay cartridgeaccording to another embodiment of the invention.

FIG. 4( b) shows a side, cross-sectional view of a reaction well.

FIG. 4( c)-1 shows a top plan view of a reaction well according to oneembodiment of the invention.

FIG. 4( c)-2 shows a top plan view of a reaction well according toanother embodiment of the invention.

FIG. 4( d) shows an end of the assay cartridge with a support tab, whichengages a propelling feature of a cartridge carriage.

FIG. 4( e) shows a front perspective view of a film piercer according toan embodiment of the invention.

FIG. 4( f) shows a side, cross-sectional view of the film piercer inFIG. 4( d) as it is used with an assay cartridge.

FIG. 4( g) shows a cross-sectional, perspective view of a cover on aportion of an assay cartridge.

FIG. 4( h) shows a top plan view of a cover on a portion of an assaycartridge.

FIG. 4( i) shows a bottom perspective view of a cover that can cover aportion of an assay cartridge.

FIG. 4( j) shows a number of side, cross-sectional views of reactionwell embodiments. A microtip is shown with each reaction well design.

FIG. 5( a) shows a top perspective view of a reaction vessel accordingto an embodiment of the invention.

FIG. 5( b) shows an exploded view of a reaction vessel according to anembodiment of the invention.

FIG. 5( c) shows a perspective cross-section view of an embodiment ofthe invention.

FIG. 5( d) shows a perspective view of a reaction vessel according toanother embodiment of the invention.

FIG. 6( a) shows a perspective view of a millitip according to anembodiment of the invention.

FIG. 6( b) shows cross-sectional view of a mounting aperture of amillitip.

FIG. 6( c) shows a portion of a millitip secured to a pipettor mandrel.

FIG. 7( a) shows a top perspective view of a cartridge loading unit.

FIG. 7( b) shows a partial top perspective view of a cartridge loadingunit.

FIG. 7( c) shows a perspective view of a cartridge loading unitpresentation lane.

FIG. 8( a) shows a front perspective view of a reagent storage unitaccording to an embodiment of the invention.

FIG. 8( b) shows a front perspective view of a portion of the reagentstorage unit.

FIG. 8( c) shows an interior of a distal wall 148 of a reagent storageunit.

FIG. 8( d) shows a front perspective view of a reagent storage unitaccording to another embodiment of the invention.

FIG. 8( e) shows a portion of a front perspective view of a reagentstorage unit according to another embodiment of the invention.

FIG. 8( f) shows a side, perspective, cross-sectional view of a reagentstorage unit.

FIG. 8( g) another side, perspective, cross-sectional view of a reagentstorage unit.

FIG. 8( h) shows a perspective, cross-sectional view showing a rearportion of a reagent storage unit.

FIG. 8( i) shows a portion of a reagent storage unit cover as itinterfaces with a containment feature of a reagent pack 400.

FIG. 9( a) shows a top perspective view of a portion of a reagent pack.

FIG. 9( b) shows an exploded view of a reagent pack according to anembodiment of the invention.

FIG. 9( c) shows an end portion of a reagent pack.

FIG. 9( d) shows a top perspective view of a barrier lid.

FIG. 9( e) shows a cross-sectional view of an end of a reagent pack.

FIGS. 10( a) and 10(b) disclose an assay cartridge in processing lane.

FIG. 10( c) discloses an assay cartridge in a heating lane.

FIG. 10( d) shows a perspective view of a processing lane heater.

FIG. 10( e) shows a front view of a processing lane heater.

FIG. 11 shows a side view of another lane heater embodiment.

FIG. 12( a) shows a microtip on a pipettor mandrel.

FIG. 12( b) shows a perspective view of a microtip.

FIG. 12( c)-1 shows a perspective view of a microtip with ventingfeatures.

FIG. 12( c)-2 shows a side view of another microtip embodiment.

FIG. 13( a) shows a microtip storage unit.

FIG. 13( b) shows a portion of a microtip storage unit.

FIG. 13( c) shows a plan view of a portion of a microtip storage unit.

FIG. 13( d) shows an exploded view of a microtip rack.

FIG. 13( e) shows a microtip rack.

FIG. 13( f) shows a rack clasp in a microtip storage unit.

FIG. 14( a) shows components in a waste lane.

FIG. 14( b) shows a hydropnuematic assembly.

FIG. 14( c) shows a perspective view of a waste lane.

FIG. 14( d) shows a perspective view of a transfer shuttle.

FIG. 14( e) shows a close up perspective view of a transfer shuttle.

FIG. 14( f) shows a front view of a processing lane.

FIG. 14( g) shows another transfer shuttle according to anotherembodiment of the invention.

FIG. 15( a) shows an XYZ transport device.

FIG. 15( b) shows an XYZ transport device Y axis arm.

FIG. 15( c) shows a Z axis elevator for the XYZ transport device.

FIG. 15( d) shows an X′ axis.

FIG. 15( e) shows a sensor system according to an embodiment of theinvention.

FIG. 16( a) shows a side, perspective view of a thermal cycler module.

FIG. 16( b) shows a side, cross-sectional view of a thermal cyclermodule.

FIG. 16( c) shows a garage with a plurality of thermal cycling cells.

FIG. 16( d) shows a thermal cycling shutter.

FIG. 16( e) shows a perspective view of a portion of a thermal cyclermodule with the shutter in a closed position

FIG. 16( f) shows an internal side view of a portion of a thermal cyclermodule with the shutter in an open position while a correspondingslidable lid is in a closed position.

FIG. 16( g) shows an internal side view of a portion of a thermal cyclermodule with the shutter in a closed position, while the correspondingslidable lid is in an open position.

FIG. 16( h)-1 shows a partial internal perspective view of internalcomponents of a slidable lid.

FIG. 16( h)-2 shows a side, perspective view of internal components of aslidable lid.

FIG. 16( i)-1 shows a side, cross-sectional view of a slidable lid in athermal cycler module, where the slidable lid is in a closed position.

FIG. 16( i)-2 shows a side, cross-sectional view of a slidable lid in athermal cycler module, wherein the slidable lid is in an open position.

FIGS. 16( j)-16(m) show a gripping feature that is configured tomanipulate a slidable lid.

FIG. 16( n) shows a side, cross-sectional view of an excitation opticsassembly, in position beneath a thermal block.

FIG. 16( o) shows a side perspective view of a thermal block.

FIG. 16( p) shows a top view of a thermal block.

FIG. 16( q) shows a side, cross-sectional view of emission andexcitation optics spring latches, as they can hold emission andexcitation optics assemblies.

FIG. 17( a) shows a block diagram of some components in a thermal cyclermodule.

FIG. 17( b) shows a graph of temperature signals vs. time from differentthermal cyclers.

FIG. 17( c) shows another graph of temperature signals vs. time fordifferent thermal cyclers.

FIG. 17( d) shows a flowchart illustrating a method according to anembodiment of the invention.

FIG. 17( e) shows an example of temperature signals produced in responseto calibrated voltage signals.

FIG. 18( a) shows a detection optics block diagram.

FIG. 18( b) shows a detection optics light path.

FIG. 18( c) shows a detection optics assembly.

FIG. 19 shows a process flow diagram illustrated methods according toembodiments of the invention.

FIG. 20( a) shows an embodiment of a cartridge heater.

FIG. 20( b) shows a section of an embodiment of a cartridge heater.

FIG. 20( c) shows an embodiment of a cartridge heater in an openposition.

FIG. 20( d) shows an embodiment of a cartridge heater in a closedposition.

FIG. 20( e) shows a section of an embodiment of a cartridge heater.

FIG. 20( f) shows components of a cartridge heater.

FIG. 20( g) shows an embodiment of an assay cartridge that may be usedwith a cartridge heater.

FIG. 20( h) shows a top plan view of a layout of the components of aninstrument according to an embodiment of the invention.

FIG. 20( i) shows an embodiment of a cartridge-swapping process.

FIG. 20( j) shows an embodiment of a lane with a lane heater.

FIG. 20( k) shows a section of an embodiment of a lane with a laneheater.

FIG. 21 shows a diagram illustrating parts of a general purpose computerapparatus.

DETAILED DESCRIPTION

PCR or “Polymerase Chain Reaction” refers to a method used to amplifyDNA through repeated cycles of enzymatic replication followed bydenaturation of the DNA duplex and formation of new DNA duplexes.Denaturation and renaturation of the DNA duplex may be performed byaltering the temperature of the DNA amplification reaction mixture. Realtime PCR refers to a PCR process in which a signal that is related tothe amount of amplified DNA in the reaction is monitored during theamplification process. This signal is often fluorescence. However, otherdetection methods are possible. In an exemplary embodiment, a PCRsubsystem takes a prepared and sealed reaction vessel and performs acomplete real-time polymerase chain reaction analysis, thermal cyclingthe sample multiple times and reporting the intensity of emittedfluorescent light at each cycle.

A “preparation location” can include any suitable location orcombination of locations which can prepare a sample for analysis.Preparation locations can include one or more of a sample presentationunit, a sample pipettor, and various processing lanes.

A “cartridge guide” can include any suitable structure for guiding anassay cartridge. In some cases, it can include a generally linearstructure to guide the assay cartridge in a linear path.

An “analysis location” can refer to any suitable location or combinationof locations where samples are analyzed.

A “processing location” can be a location where samples are processed. Aprocessing location can be within a preparation location. For example, aprocessing location can have a plurality of processing lanes that canprocess a sample.

A “reagent storage unit” may refer to a unit that is configured to storereagents.

A “reagent pack” may include any suitable container that can store areagent. An example of a reagent pack can include a generallyrectangular elongated body formed to include multiple reagentreceptacles including one or more large reagent receptacles, and one ormore relatively smaller reagent receptacles, as well as features tofacilitate handling and automation.

A “processor” may comprise any suitable data processing device that canbe used to process data. Such processors may include one or moremicroprocessor working together to process data and provideinstructions.

A “controller” may also be a data processing device that can be used toprocess data or provide control functions. A controller may include oneor more microprocessor, or it could be a general purpose computer insome embodiments.

A. Overall System Layout

An automated instrument for the determination of nucleic acids accordingto an embodiment of the present invention is shown in FIG. 1( a),designated by reference number 100. As shown in FIG. 1( a), oneembodiment of the instrument of the invention includes a generallyrectangular housing 102 with sides defining the front, back, left andright sides, top and bottom as illustrated. The automated instrument canbe a single, enclosed system, and can include a horizontal working deckthat incorporates readily accessible areas 110 for an operator to addsamples for analysis and consumables for use in processing the samples.It also includes a data entry device 106 and a display 108. Embodimentsof the invention include a fully automated, random access system fordetermining specific nucleic acid sequences in samples. The systemincludes consumables incorporating necessary reagents for performance ofa variety of assays, reaction sites, and transfer devices. Sufficientstorage space for consumables is provided on the system to permit it torun with minimal operator intervention for an extended time.

The system can combine two functions: sample preparation in the form ofisolation of nucleic acids from the sample matrix, and detection ofspecific sequences within these isolated nucleic acids. Towards thisend, the system can have at least two distinct functional areas: oneincluding instrumentation to process samples using the consumables and asecond including the instrumentation and reagents for nucleic acidamplification and detection. The system also includes holders forsamples, containers for wastes, and connections for power andinformation. These are integrated in a single unit to provide a systemthat performs major functions of sample handling, nucleic acidisolation, and amplification and detection, plus supporting functions ofsupply and consumable management, information management, andmaintenance. In some embodiments, to support sample throughput whileretaining scheduling flexibility, the sample preparation portion of thesystem processes samples in a sequential fashion as they enter thesystem while the detection portion of the system performs amplificationand detection in parallel.

Combining these functions into a single, highly automated, selfcontained system provides seamless integration of molecular diagnosticsinto the workflow of the clinical laboratory. A further purpose is toperform all steps of nucleic acid determination to produce clinicallyacceptable results without the need for user intervention. The systemadvantageously allows users to load samples as they become available,and to perform determinations on those samples as dictated by the needsof the patient and their physician, without constraints on sample oranalyte order being imposed by the system.

FIG. 1( b) shows a plan view of the embodiment of FIG. 1( a) from above,with some components removed to clarify the basic structural andfunctional modules. FIG. 1( b) also shows three distinct locationsincluding an analysis location 96 where sample analysis can occur, and apreparation location 98 where the sample can be prepared for analysis.FIG. 1( b) also shows three distinct locations including an analysislocation 96 where sample analysis can occur, a preparation location 98where the sample can be prepared for analysis, and a material storagelocation 92 where preparation and analysis materials can be stored. Thethree illustrated locations can be adjacent to each other.

The system shown in FIG. 1( b) can be used to perform a variety ofmethods, including a method comprising providing an assay cartridgecomprising a first compartment and a second compartment with a cartridgeguide, transferring a first reagent from a first compartment to a secondcompartment in an assay cartridge using a first pipettor at apreparation location, and transferring a second reagent from a reagentpack in a reagent storage unit or in a materials storage location to thesecond compartment using a second pipettor.

The system may include an instrument, which may include a samplepresentation unit 110 for loading samples, a sample pipettor 70 fortransferring samples, a cartridge loading unit 112 for loadingdisposable assay cartridges onto the system, a reagent storage unit 10for storing reagents, a set of processing lanes 116 for processingsamples, a transfer shuttle 50 for transferring assay cartridges, an XYZtransport device 40 for transferring materials, a microtip storage unit20 for storing disposable pipette tips, a collection of thermal cyclermodules 30 for amplification, and an optical detector (not shown) fordetection of products from the detection reaction. The XYZ transportdevice 40 may include an XYZ gantry, as well as an XYZ pipettor. Theprocessing lanes may be present in the preparation location 98.

The gantry can perform a number of functions. For example, it can beconfigured to: position the mandrel to remove the vessel plug from thesecond compartment; position the mandrel to mate the vessel plug to thevessel base in the first compartment; position the actuator to move thelid from the closed position to the open position; position the mandrelto seat the amplification vessel in the block; and position the actuatorto move the lid from the open position to the closed position.

The system can include processing lanes 116 that perform the operationalsteps needed for nucleic acid extraction and purification from abiological or patient sample. Each processing lane 116 can accommodatean assay cartridge 200. When the system uses a linearly arranged assaycartridge 200 each processing lane may extend linearly relative to thelong axis of the assay cartridge. Such processing lanes 116 may reflectthe dimensions of the assay cartridge 200, reducing the need to orientthe assay cartridge and permitting the system to package multipleprocessing lanes in a space-efficient parallel manner. In someembodiments, the system includes processing lanes that are physicallyarranged in an order approximating their order of use in at least someprotocols. This advantageously minimizes the distance and time thesystem needs to transfer assay cartridges between processing lanes.Alternatively, the system may include processing lanes with similarfunctions grouped together. This advantageously minimizes the time spentperforming repetitive functions, such as, for example, washing.

As shown in FIG. 1( b) the system may include different types ofprocessing lanes that support functions appropriate to differentprocessing steps. In some embodiments, the system includes replicates ofsome lane types, allowing processing of multiple assay cartridges 200 inparallel. Examples of processing lane types include a cartridge loadinglane 116(f), a transfer lane 50, a heated temperature stabilization lane116(j), a wash lane 116(a) and 116(b), an elution lane 116(e), anamplification preparation lane 116(g), and a waste lane 116(c). In someembodiments, the system includes 13 processing lanes in the followingsequence:

LANE POSITION LANE TYPE 1 AMPLIFICATION PREPARATION LANE 2 CARTRIDGELOADING LANE 3 ELUTION LANE 4 WASTE LANE 5 HEATED TEMPERATURESTABILIZATION LANE 6 AMBIENT TEMPERATURE STABILIZATION LANE 7 AMBIENTTEMPERATURE STABILIZATION LANE 8 WASH LANE 9 WASH LANE 10 WASH LANE 11WASH LANE 12 WASH LANE 13 WASH LANEThe first lane position can be near the center of the instrument, withsuccessive lanes numbered toward the right side of the system as viewedfrom the front. Successive lane positions may be disposed adjacent thepreceding lane position. Alternatively, the system may incorporate oneor more processing lanes that individually incorporate all of theprocessing tools needed to perform each processing step.

In some embodiments, the instrument includes an area for connecting to alaboratory automation device 80 for automated delivery of samples from acentral location in the laboratory. A conventional instrument frameworkprovides physical and operational support to these modules. Theframework provides support components, including electrical powersupplies; airflow control components such as fans, blowers, ducts fordirecting airflow, and air filters; and communications and controlcomponents such as displays, one or more control computers, wiring, andother interconnects. The sections below describe each of the basicstructural and functional modules in more detail.

FIG. 1( c) shows a detailed top view of an embodiment of the instrument,with some components removed for clarity. The components shown in FIGS.1( a)-1(c) are described in greater detail below.

The system according to an embodiment of the invention can include apreparation location 98 to process a sample. The preparation location 98can be any suitable location where sample preparation may take place. Insome embodiments, the preparation location is found on the right side ofthe instrument when facing the front.

The preparation location 98 can include a sample presentation unit 110where samples are loaded onto the system, a set of processing lanes 116where sample preparation takes place and a sample pipettor 70 fortransfer of sample to an assay cartridge for processing. The assaycartridge can be transferred in the system and the preparation location98 using a cartridge guide (which is described in further detail below).Samples are prepared for amplification in a disposable assay cartridgethat includes a first compartment and a second compartment. In someembodiments, the second compartment may be a reaction well, while thefirst compartment may be a small, medium, or large reagent well. Theprocessing lanes 116 can include features to retain, warm, and guide theassay cartridge, and a first pipettor configured to transfer liquidsfrom at least the first compartment to the second compartment.

The system can also include a reagent storage unit 10 that is configuredto store at least one reagent pack. In some cases, the reagent storageunit comprises a plurality of reagent packs for storing reagents forperforming a PCR process. In some embodiments of the invention, thereagent storage unit 10 and microtip racks 120 can be in the materialsstorage location 92. A second pipettor (not shown) can be associatedwith the XYZ transport device 40 and can be disposed to travel betweenthe reagent storage unit 124 and the materials storage location 92 underthe direction of a central controller 94 for the system.

The central controller 94 can direct the operation of any of thecomponents described herein by providing instructions to varioussub-controllers within the system. The central controller 94 can includeany of the components shown in FIG. 20 (which describes a computerapparatus).

The central controller 94 can be operatively coupled to the firstpipettor and to the second pipettor and is configured to direct thefirst pipettor to transfer a first reagent from the first compartment(e.g., a small, medium, or large reagent well) of the assay cartridge tothe second compartment and to direct the second pipettor to transfer asecond reagent from the reagent pack to the second compartment (e.g., areaction well). Suitable examples of first and second reagents (e.g.,wash fluids, buffers, etc.) are provided below. The use of a firstpipettor and a second pipettor advantageously permits the system toquickly and accurately dispense both large and small volumes to theassay cartridge, by avoiding the risk of inaccuracy due to attempting totransfer small volumes using a large volume pipettor and the risk ofinaccuracy due to attempting to deliver large volumes through repeateddispenses using a small volume pipettor. This operational flexibilitysupports both the processing or relatively large sample volumes and theuse of compact reagent packs that store concentrated reagents.

Following the completion of sample preparation, the treated sample, plusadditional reagents, is transferred to the detection and amplificationportion of the system. The detection and amplification portion of thesystem may be at an analysis location 96. The analysis location 96 cancontain a plurality of analysis or analytical units, such as thermalcyclers, and may be positioned at any suitable location within thesystem. In some embodiments the analysis location 96 is found on theleft side of the system when facing the front. This location maximizesthe distance between the preparation portion and the amplification anddetection portions of the system. This permits the introduction ofbarriers to reduce contamination, including but not limited to directedairflow, ultraviolet light, and physical barriers such as partitions orfilters, while allowing easy access for servicing. In anotherembodiment, the detection and amplification portion of the system can beencased within the instrument housing, below the working deck.

In the embodiment shown in FIG. 1( c), amplification and detection areprovided by a bank of thermal cycler modules 30. The thermal cyclermodules within bank 30 may process samples independently butsimultaneously, with each thermal cycler module processing a singlesample at a time. Scheduling of processing in the thermal cycler modulesin bank 30 may be balanced to equalize the degree of wear betweendifferent thermal cyclers. One or more thermal cycler modules may bereserved for use in circumstances where additional modules beyond thosenecessary for normal operations are needed. Examples of such atypicalcircumstances include the failure of a thermal cycler module and theprocessing of an urgent or STAT sample. The number of these thermalcycler modules can vary between different embodiments of the invention,being optimized for the desired throughput of the system.

The need for random access processing and the possibility ofcontamination between amplification products and samples makes the useof consumables central to system operation. In some embodiments, systemconsumables include assay cartridges used for storage of selectedreagents and isolation and purification of nucleic acids from samples;reaction vessels for amplification and detection; reagent packs forstoring selected reagents; millitips for large volume pipettingoperations; microtips for small volume pipetting operations; andmicrotip racks to retain microtips.

As shown in FIG. 1( d), the system provides storage areas for spentconsumables. These storage areas may be below the working deck in orderto reduce the chances for contamination from stored waste. As describedin greater detail below, waste liquids may be stored in a designatedliquid waste container 94. Similarly, solid wastes may be temporarilystored on the system in a designated solid waste container 92. Wastecontainers may be held within enclosed cabinets in the lower portion ofthe system. These cabinets may be kept at negative pressure in order toprevent aerosols and particulates from the waste containers fromreaching the working deck of the system, and may be convenientlyaccessed in order for the user to empty the waste containers. Wastestorage areas may also include mechanisms to inactivate contaminantsfollowing inadvertent release, including ultraviolet light sources.

Another embodiment of the invention can be directed to a systemcomprising a first pipettor and a second pipettor, as well as acontroller operatively coupled to the first pipettor and to the secondpipettor. The controller is configured to direct the first pipettor totransfer a fluid from a first compartment in an assay cartridge or froma reagent pack in a reagent storage unit to a reaction vessel in theassay cartridge, and to direct the second pipettor to remove thereaction vessel from the assay cartridge. In the system, an assaycartridge comprising a first compartment and a second compartment can beguided with a cartridge guide, and a fluid (such as a processed sample)is transferred from the first compartment (which may be a reaction well)or from a reagent pack in a reagent storage unit to a reaction vessel inan assay cartridge using a first pipettor. The reaction vessel is thenremoved from the assay cartridge, and then transferred to a thermalcycler module using the second pipettor. The first pipettor can be amillitip pipettor and the second pipettor can be a microtip pipettor.Other suitable details regarding such embodiments of the invention canbe found below.

The second pipettor can advantageously have multiple uses includingtransferring fluids as well as moving reaction vessels within thesystem. Since separate devices are not needed to perform these and otherfunctions, the system according to embodiments of the invention can becompact and less complex than other types of systems.

Yet another embodiment of the invention is directed to a system, whichcan be for determining the presence of a nucleic acid in a sample. Thesystem may comprise a cartridge loading unit 112 to accept a pluralityof assay cartridges. The cartridge loading unit 112 can include astorage location to support the plurality of assay cartridges, a loadinglane coupled to the storage location, and a loading transport coupled tothe storage location and to the loading lane and configured to move anassay cartridge from the storage location to the loading lane. Thesystem can also include a plurality of processing lanes (e.g., 116(a),116(b), 116(c), 116(e), 116(g), etc.) to process an assay cartridge,each processing lane configured to operate on an assay cartridge, and ashuttle 50 to move the assay cartridge among the loading lane and theplurality of processing lanes, the shuttle positionable in alignmentwith the loading lane 116(f) and in alignment with each of the pluralityof processing lanes; and a controller 94 operatively coupled to theloading transport, to the shuttle 50, and to the plurality of processinglanes. As shown in FIG. 1( b), the processing lanes (e.g., 116(a),116(b), 116(c), 116(e), 116(g), etc.) and the loading lane 116(f) areparallel to each other, and they are all perpendicular to the travelpaths of the transfer shuttle 50.

In this embodiment, a method for using the system may comprise loading aplurality of assay cartridges into a storage location in a cartridgeloading unit, moving an assay cartridge to a loading lane using aloading transport, moving the assay cartridge to a shuttle, and movingthe assay cartridge to one of a plurality of processing lanes, eachprocessing lane configured to process the assay cartridge using adifferent process.

The particular arrangement of a loading lane and various processinglanes with an assay cartridge transport shuttle provides a number ofadvantages. In embodiments of the invention, assay cartridges can beprovided to a transfer shuttle, which can access various processinglanes as needed for particular protocols. This provides for flexibilityin processing, while providing for a compact system.

Yet another embodiment of the invention can be directed to a systemcomprising a preparation location 98 for processing samples, a reactionvessel for containing the processed sample, an analysis location 96 forcharacterizing the processed sample, and a transport device fortransferring the reaction vessel between the preparation location andthe analysis location. An example of a transport device can be the XYZtransport device 40. The system may also comprise a plurality ofnon-identical processing lanes 116 in the preparation location 98, theprocessing lanes 116 configured to perform different processingfunctions, and a plurality of identical analytical units in the analysislocation. The analytical units may comprise thermal cycler modules,which are described in further detail below.

This particular system arrangement can provide for flexibility inprocessing, while providing good throughput.

Yet another embodiment of the invention is directed to a system fordetermining the presence of a nucleic acid in a sample, the systemcomprising a first processing lane configured to perform operations on asample in an assay cartridge, a transfer shuttle 50 configured to moveassay cartridges into and out of the first processing lane, and acontroller 94 to direct operation of the system. The first processinglane would be any of the described processing lanes 116 shown in FIG. 1(b). The controller 94 can be operatively coupled to the first processinglane and to the transfer shuttle 50, and can be configured to execute afirst protocol and a second protocol. The first and second protocols caninvolve any suitable number or type of processing steps, where the firstprocessing lane is used in both protocols, where the two protocolsprocess different samples in different assay cartridges.

The controller 94, in executing the first protocol, directs the transfershuttle 50 to move a first assay cartridge into the first processinglane, and after a fixed interval, directs the transfer shuttle to movethe first assay cartridge out of the first processing lane, and withinthe fixed interval directs the first processing lane to execute a firstsequence of operations. The fixed interval may comprise any suitableamount of time. The controller 94, in executing the second protocol,directs the transfer shuttle 50 to move a second assay cartridge intothe first processing lane, after the fixed interval, directs thetransfer shuttle to move the second assay cartridge out of the firstprocessing lane, and directs the first processing lane to execute asecond sequence of operations.

The first sequence of operations can be different from the secondsequence of operations. The first and second protocols and theirsequence of operations may differ in any suitable manner. For example,the first and second protocols may include common processing steps, butmay different according to the duration processing or the parametersused for processing. For instance, in some embodiments, two differentprotocols may have similar processing steps, but the processing stepsmay differ because they are performed at different temperatures and/orfor different periods of time. In another example, two protocols mayhave similar steps, but they may be performed in different orders. Forexample, a first protocol may include steps A, B, and C performed inthat order. A second protocol may include steps B, A, and C performed inthat order. Lastly, in yet another example, different protocols mayinclude different sets of steps. For example, a first protocol maycomprise steps A, B, C, and D, while a second protocol may comprisesteps B, D, E, F, and G.

B. Sample Presentation Unit

FIG. 2( a) shows a perspective view of an embodiment of the samplepresentation unit.

FIG. 2( b) shows an embodiment of the pusher carriage.

As shown in FIG. 2( a) the sample presentation unit 110 can havemultiple functions related to handling of samples to be analyzed on thesystem. The sample presentation unit 110 may act as a buffer between theuser and the instrument, providing a holding area for storage of sampleswhen they are not being actively processed by the instrument. The samplepresentation unit 110 may also provide a mechanism for presenting thesamples or volumes taken from the samples to processing portions of theinstrument. A user may place samples onto the sample presentation unit110 as they become available in the laboratory; the instrument maysubsequently access the loaded samples as its processes require. Thisbuffering mechanism advantageously incorporates the system into thelaboratory workflow by integrating the essentially random appearance ofsamples requiring testing with the system's scheduled timingrequirements.

One embodiment of the sample presentation unit 110 processes samplespresented in sample holders 616. The sample presentation unit 110 mayinclude, among other components, a sample base 602, an input queue 628,an output queue 640, a presentation carriage 634, and a sample barcodereader 622. The sample presentation unit 110 may include a sample returnlane for routing samples from the output queue 640 back to the inputqueue 628. This arrangement supports secondary testing of specificsamples as designated by the system in response to the results of theinitial test, also known as reflex testing. Such a secondary test may bea repeat of the initial test (for example, in response to a reportederror condition) or a different test. In some embodiments, the samplepresentation unit may include a barcode reader for recording sampleinformation prior to placing a sample on the system. Such a barcodereader may be a hand held unit. In an alternative embodiment, the samplepresentation unit may have a vertical arrangement, with input and outputqueues comprised of elevator assemblies that carry samples into thesystem for analysis and out of the system for removal, respectively.

In some embodiments, the sample presentation unit 110 may accept samplesin a variety of containers in the form of sample tubes. Sample tubes maybe of several different types that differ by size, by type of sample, orby some other attribute or some combination of attributes. Examples ofsample tubes are primary blood collection tubes, swab collection tubes,swab culture tubes, secondary cups and tubes containing samplesaliquoted from primary tubes. These samples presented in these sampletubes may include but are not limited to blood, serum, plasma, spinalfluid, saliva, urine, tissue samples, and fecal specimens. Samples mayalso include purified or partially purified materials generated byprocessing of specimens prior to presentation to the system. In additionto samples, sample tubes may also contain swabs and other samplecollection devices that are utilized in taking surface samples fromwounds and other test areas. Such sample tubes may include a barcode orother machine-readable indicia that designates the patient from whichthe sample originated, sample type, testing to be performed, or otherinformation. This information may be entered into the system via asuitable reader prior to or after loading the sample onto the system, Insome embodiments of the invention, the user loads samples onto thesystem as individual tubes. In other embodiments, the user may loadsamples onto the system as individual tubes that are held in sampleholders 616.

Sample holders 616 may accommodate a plurality of sample tubes. Thisadvantageously reduces user effort by reducing the number of loading andunloading operations required, since each operation may involve multiplesamples. The use of sample holders 616 additionally reduces the level ofuser attention required to operate the system as sample holders 616 maybe self-supporting, whereas individual sample tubes typically are not.This is useful to prevent accidental spills, which reduces the chancesof contamination and preserves sample integrity. In addition, somesamples, such as whole blood tubes treated to separate cells from plasmaor serum, may generate erroneous results if tilting or dropping re-mixesthe contents.

Sample holders 616 may be any of a variety of forms including disks,rings, sectors, or linear racks. In some embodiments, the sample holders616 are linear racks with support tabs at either end to maximize packingdensity. In some embodiments of the invention the sample holders 616 arein the form of linear racks that hold four sample tubes, such as thatshown in FIG. 2A. Users can easily manipulate these sample holders 616with one hand, and specialized centrifuge rotors permit centrifugationof sample tubes while held in such sample holders 616. In an alternativeembodiment, sample holders may be loaded into the sample presentationunit while held in a rack that supports multiple sample holders. In yetanother embodiment, sample holders may be loaded into the samplepresentation unit while held in a device that joins multiple rackstogether. In another embodiment, the assay cartridge 200 may include afeature that supports a sample tube, thereby also serving as a singleposition sample tube holder.

The sample base 602 may support and provide connection points for othercomponents of the sample presentation unit 110. In some embodiments, thesample base 602 is an essentially planar surface disposed horizontallybeneath the other components of the sample presentation unit 110. Thesample base 602 can define the bottom of the sample presentation unit110. In some embodiments, the sample base 602 is “T-shaped,” with arelatively narrow stem 626 joining near the midpoint of andperpendicular to a broader crossbar 608. This stem 626 can support thepresentation track 624 and the presentation carriage 634 that rides onthe presentation track 624. The stem 626 may project in an inwarddirection, toward the rear of the system.

The crossbar 608 can support the input queue 628 and the output queue640. One terminus of the crossbar 608 defines both the entrance pointand the entrance direction for sample holders 616 onto the system. Theopposite terminus can define the exit point and the exit direction forthe sample holders 616.

The input queue 628 can serve as a storage location for one or moresample holders 616 containing samples that have not yet been processed.In some embodiments, the input queue 628 can hold up to 12 (or more)sample holders 616. The input queue 628 can support the sample holders616 in an ordered arrangement such that the instrument processes thesample holders 616 sequentially, as loaded by the user. Thisadvantageously allows a user to determine the order of processing bysimply loading sample holders 616 onto the input queue 628 in thedesired order. In some cases, users may load samples of higher priorityfirst. In some embodiments, the input queue 628 may have a temporaryholding area and an onload queue that feeds sample holders into thesystem. This arrangement permits the system to alter the loadingsequence of the sample holders by temporarily diverting one or moresample holders from the onload queue into the temporary holding area,reinserting the diverted sample holders into the onload queue at a latertime in order to prioritize samples. In an alternative embodiment, theinput queue may include a dedicated position for onloading of one ormore high priority or STAT samples. In some embodiments, the input queue628 includes an input support, an input spill tray 620, and a pusherplate 617.

The input support can be a portion of the crossbar 608 of the samplebase 602 that extends from near the entrance end of the samplepresentation unit 110 to near the junction of the stem 626 and crossbar608. The input support can include a pair of support rails arrangedparallel to one another at a separation distance corresponding to thedistance between support tabs disposed at opposite ends of sampleholders 616. The support rails can define the boundaries of the activeregion of the input queue 628 and can connect to the sample base 602. Inoperation, sample holders 616 can rest on the support rails, and may befree to slide along the support rails with sample holders 616 loadedearlier in the process pushed along the support rails by adjacent sampleholders 616 loaded later. In alternative embodiments, sample holders 616may be moved by resting the sample holders upon a moving belt or a setof drive wheels.

The input spill tray 620 may lie between and beneath the support rails,and serves to control contamination by containing any spills, drips, orleakage from sample tubes. The input spill tray 620 can be an oblong oressentially rectangular structure, and can include a floor withcontainment walls on two sides and the entrance end. The input spilltray 620 may be open at the top and at the exit end of the input queue628. In some embodiments, the floor includes a deeper sump region nearthe entrance end. The floor may slope towards this sump region toprovide collection and containment of spilled liquids at one locationfor easy removal. The input spill tray 620 can be removable, and mayrest on other sample presentation components including the sample base602.

The queue pusher may include a pusher carriage 612 that includes apusher plate 617 to push against the sample holder 616 closest to theentrance end of the input queue 628, a queue track 654 to guide themovement of the pusher carriage 612, and a queue drive 614 to move thepusher carriage 612 along the queue track 654. The system may include afunction whereby the user can signal the system to move the queue pusheraway from the terminal sample holder, allowing the user manipulate thesample holder queue to load a sample holder including sample tubescontaining STAT or urgent samples at the front of the sample holderqueue, for early presentation to the system on reengagement of the queuepusher. In one embodiment, the input queue may include a STAT queuepusher that manipulates that sample holder queue to permit a user toload a sample holder that includes sample tubes containing STAT orurgent samples. In an alternative embodiment, the queue pusher caninclude grippers that clasp the terminal sample holder, allowing thequeue pusher to manipulate the sample holder queue to permit a user toload a sample holder that includes sample tubes containing STAT orurgent samples.

The pusher carriage 612 may include one or more bearings 650 to engagethe queue track 654, and a pusher plate 617 to engage the flat side ofthe last sample holder within the input queue 628, and a bracket 652 toconnect the pusher plate 617 to the bearing 650. The pusher plate 617pushes against the last sample holder in the queue, which in turn pushessuccessive sample holders 616, if any are present, to move all loadedsample holders 616 toward the exit end of the input queue 628.

As shown in FIG. 2( a) and FIG. 2( b), the pusher plate 617 may be aplanar sheet oriented vertically within the input spill tray 620, andcan extend across most of the width of the input spill tray 620. Thebracket 652 may include an upper horizontal member 646, a verticalmember 648, and a lower horizontal member 649. The upper horizontalmember 646 extends from the pusher plate 617 above the closed end wallof the input spill tray 620. The vertical member 648 extends from anedge of the upper horizontal member 646 to below the level of the inputspill tray 620. The lower horizontal member extends from the lower edgeof the vertical member 648 toward the queue track 654 and couples to thebearing 650. A portion of the bracket 652 may ride within a gap betweenthe input spill tray 620 and one of the support rails. This arrangementadvantageously allows the pusher plate 617 to move within the innerspill tray without requiring an opening in the inner spill tray. Anabsence of openings in the inner spill tray helps in the containment ofspills and reduces possible contamination.

The queue track 654 may extend under the input queue 628 along thecrossbar 608 of the sample base 602. The queue track 654 is fixed to thesample base 602 and guides the motion of the pusher carriage 612 alongthe pusher motion path. The queue track 654 connects to the pushercarriage 612 through complementary bearings 650. In some embodiments,the queue track 654 is a linear guide rail and the bearings 650 arecaged ball bearing blocks or caged roller bearing blocks.

The queue drive 614 may move the pusher carriage 612 along the queuetrack 654 by any of a number of drive methods, including the use of alead screw and nut, a linear motor, or a pneumatic actuator. In someembodiments, the instrument uses a motor that is attached to the samplebase 602 near an end of the queue track 654, and is coupled to a drivepulley. An idler pulley may be attached to the sample base 602, oralternatively to the support rails, near the opposite end of the queuetrack 654 by an attachment that permits adjustment of the separationbetween the idler pulley and the drive pulley. A timing beltsubstantially parallel to the queue track 654 runs from the drive pulleyto the idler pulley and couples to the pusher carriage 612. Adjustmentof the separation between the idler and drive pulleys permits adjustmentof the force applied to the sample holders 616 via the pusher plate 617.Rotation of the motor drives the timing belt and moves the pushercarriage 612 along the queue track 654.

As noted above, in some embodiments of the invention, sample tubes maybe transported onto the system through an input queue individually,without the use of a sample holder. In such an embodiment, the inputqueue can utilize individual pucks that each support one sample tube;such pucks may be impelled using a magnetic drive. Alternatively,individual tubes may be transported using a belt drive or a set of drivewheels. In other embodiments, the input queue may include a storagelocation for holding individual sample tubes, which are transported ontothe system using a pick and place device. Such an embodimentadvantageously simplifies prioritization of sample testing by the systemby permitting it to select sample tubes independent of order in whichthey are loaded by the user.

The output queue 640 is a storage location for sample tubes followingremoval of an aliquot that is utilized for testing purposes. The outputqueue may also serve as a site for the offloading of sample aliquotsthat have been processed by the system, for retrieval by the user forfurther testing. In some embodiments, the output queue 640 supports thesample holders 616 in an ordered arrangement similar to that of theinput queue 628. The output queue 640 may include an output support 638and an output spill tray 604.

The output support 638 extends from near the area where the stem 626 andcrossbar 608 of the sample base 602 join to near the exit end of thesample presentation unit 110. The output support 638 may be similar instructure and function to the input support. In some embodiments, theoutput support 638 includes parallel support rails, one of which may becontiguous with one of the support rails of the input support. In someembodiments, sensors mounted to one of the parallel support rails mayindicate when the output queue 640 has reached a predetermined filllevel. These sensors may be optical sensors.

The output spill tray 604 may be similar in form to the input spill tray620 and performs a similar function. However, it is supported by theoutput support 638. The output spill tray 604 rests within the outputsupport 638 in an approximately reverse orientation to that of the inputspill tray 620, with the end vertical wall oriented towards the exit endof the sample presentation unit 110. The sump of the output spill tray604 may therefore be near the exit end, with the open end of the outputspill oriented towards the entrance end. This advantageously creates anopen path for sample holders 616 to travel either directly or indirectlyfrom the input queue 628 to the output queue 640. The manufacturingprocess may employ any of a variety of methods to form the input andoutput spill trays 604. In some embodiments, the spill trays are vacuumformed plastic.

The input queue 628 and the output queue 640 may be aligned with oneanother, separated by a gap that is approximately the width of a sampleholder. A presentation shuttle 656 can intrude within this gap andextend toward the inboard end of the sample base 602.

The presentation shuttle 656 transports sample holders 616 along asample motion path that extends over several operative positions. Thissample motion path may be oriented transverse to the path of the samplepipettor 700. Operative positions may include a transfer position 642, asample identification position 644, and an aspiration position 632. Thesample identification position 644 may be disposed between the transferposition 642 and the aspiration position 632.

The transfer position 642 can be disposed within the aforementioned gapbetween the input queue 628 and the output queue 640. The aspirationposition 632 can be disposed near the inboard end of the sample motionpath, where the sample motion path intersects the path of the samplepipettor. The sample identification position 644 may be disposed betweenthe transfer position 642 and the aspiration position 632 and is alignedwith a sample reader 622.

The presentation shuttle 656 may include a presentation carriage 634 toengage the sample holder 616, a presentation track 624 to guide motionof the presentation carriage 634, a presentation drive to move thepresentation carriage 634 along the presentation track 624, anaspiration channel 630 to support the sample holder during aspiration, asample gate 606 to prevent unintended movement of sample holders 616,and a faring 636 to protect the sample holders 616 and reducecontamination.

The presentation carriage 634 engages a controlled surface of a sampleholder in order to move the sample holder within the cartridge guide. Insome embodiments, the controlled surface is an essentially vertical edgeof the sample holder that is disposed inward of one of the support tabs.In some embodiments, the presentation carriage 634 is a narrow“U-shaped” body that includes a pair of vertical members and aconnecting base member. The base member may include one or more bearings(not shown) to connect the presentation carriage 634 to the presentationtrack 624. The vertical members may rise from either end of the basemember and terminate in short vertical protrusions that engage thesupport tabs of the sample holders 616. The U-shaped body may have awidth approximating that of a sample holder. When positioned within thetransfer position 642 between the input queue 628 and the output queue640, the presentation carriage 634 effectively joins the input supportto the output support 638 as one continuous path. The gaps between thepresentation carriage 634 and the input support between the presentationcarriage 634 and the output support 638 may be narrower than the widthof a sample holder. As a result, when the presentation carriage 634 iswithin this transfer position 642, motion of the sample pusher (617) maypropel a sample holder smoothly from the input queue 628 to thepresentation carriage 634 while simultaneously propelling a differentsample holder held within the presentation carriage 634 to the outputqueue 640. Cooperative motion between the sample pusher and thepresentation carriage 634 serves to load and unload sample holders 616onto the presentation carriage 634 and may also serve to transfer sampleholders 616 directly from the input queue 628 to the output queue 640without intervening movement along a sample motion path associated withprocessing and analysis of the sample. In an alternative embodiment, theoutput queue may have a dedicated drive mechanism for offloading sampleracks from the transfer position.

The presentation track 624 may extend along the stem 626 of the samplebase 602 and defines the sample motion path. The presentation track 624may be fixed to the stem 626 of the sample base 602, and guides motionof the presentation carriage 634 along the sample motion path. Thepresentation track 624 may connect to the presentation carriage 634through complementary bearings 650. In some embodiments, thepresentation track 624 is a linear guide rail and the bearings are cagedball bearing blocks or caged roller bearing blocks.

The presentation drive moves the presentation carriage 634 along thepresentation track 624, and may do so by any of a number of drivemethods. Such drive methods include but are not limited to a lead screwand nut, a linear motor, or a pneumatic actuator. In some embodiments,the instrument uses a motor attached to the sample base 602 near one endof the presentation track 624 and coupled to a drive pulley. An idlerpulley may be attached to the sample base 602 near the opposite end ofthe presentation track 624, by an attachment that allows adjustment ofthe separation between idler pulley and drive pulley. A timing beltsubstantially parallel to the presentation track 624 may run from thedrive pulley to the idler pulley couple to the presentation carriage634. The tension of this timing belt may be altered by adjusting theseparation between the idler pulley and the drive pulley. Rotation ofthe motor drives the timing belt and impels the presentation carriage634 along the presentation track 624.

In some embodiments, the aspiration channel 630 may be a rectangulartunnel. The length of the aspiration channel 630 can approximate thelength of a sample holder with a width slightly greater than the widthof the sample holder. The aspiration channel 630 lies along the samplemotion path and may extend beyond the aspiration position 632. Anopening in the upper surface of the aspiration channel 630 at theaspiration position 632 gives access to the sample pipettor (not shownin FIG. 2( a)). This arrangement of the aspiration channel 630advantageously supports a sample holder in a defined position that isconsistent for each sample tube in the sample holder and necessary foraccurate pipetting.

The aspiration channel 630 may include one or more sample springs toimpel a sample holder that is within the aspiration channel 630 againstan internal aspect of the aspiration channel 630 in order to bettercontrol lateral and vertical position. Sample springs may be strips of arelatively stiff but elastic material, such as spring steel, mounted toan aspiration channel 630 wall. Alternatively, sample springs mountedwithin the presentation lane at the aspiration position may be used tostabilize lateral and vertical position of the assay cartridge withoutan aspiration channel.

The sample gate 606 may be a generally “L-shaped” member approximatelythe same width as the presentation carriage 634, and may have a roundedfree end. The opposite arm of the L may mount to the sample base 602near the transfer position 642. The mounting may connect the member tothe sample base 602 through a pivot near the end of the arm. This pivotmay include a spring. When the presentation carriage 634 is outside ofthe transfer position 642, the sample gate 606 pivots to occupy at leasta portion of the transfer area, thereby preventing movement of a sampleholder into the transfer area. The presentation carriage 634, uponreturning to the transfer position 642, impacts the rounded free end ofthe sample gate 606 to push the sample gate 606 out of the transferposition 642. This arrangement advantageously allows loading of theinput queue 628 while the presentation carriage 634 is engaged in sampletransfer operations. This has the beneficial effect of furtherdecoupling scheduled instrument operations from user action, freeing theuser to load and unload samples without concern for instrument timing.

In some embodiments, the shape of the sample gate 606 can be anapproximately rectangular shape, oriented with the long axis vertical. Alarge crescent-shaped section can be removed that extends from the topright corner to the middle of the lower short edge. It may becharacterized as a modified “C,” rather than an “L” shape as shown inFIG. 2( a).

In some embodiments, a protective faring 636 may cover the sample motionpath to prevent contamination of the sample tubes. The faring 636 may bea plastic or sheet metal shield formed to extend above and on the sidesof sample holders 616 as they pass along the sample motion path. Thefaring 636 may include openings to allow access to the sample pipettorand to the sample reader 622.

The sample presentation unit 110 may also have of a sample reader 622 toidentify individual sample tubes as they enter the system by reading aunique sample identification associated with each sample tubes. Sampleidentification typically includes a form of machine readableinformation, such as a barcode or other graphical code. Well-establishedpractice surrounds use of such codes in clinical laboratories.

In some embodiments, the sample reader 622 is an image-based or scanningbarcode reader positioned along the sample motion path at the sampleidentification position 644. The sample reader 622 is oriented so thatthe scanner has a view of any sample identification labels affixed tosample tubes or sample holders 616 as the sample holders 616 aretransported on the presentation shuttle 656. The sample reader 622 mayconnect to the instrument controller in order to pass sampleidentification information to the instrument controller. The instrumentcontroller, in turn, may query off-board computer systems or an on-boarddatabase to determine which assay or assays are to be performed on theidentified sample.

The sample presentation unit 110 may include a sample cover. The samplecover controls user access to the input queue 628 and the output queue640, and may also serve to reduce contamination and sample evaporation.The sample cover may include a mechanized latch and at least one controlswitch. The sample cover may be at least partly transparent in order toallow users to gauge the extent of work in progress and occupancy of theinput and output queues 640.

The sample presentation unit 110 may also include one or more covers. Inone embodiment, the sample presentation unit 110 may have a hinged orsliding cover to protect the sample tubes. Further, the cover could belatched when the sample presentation unit 110 is in operation.

In some embodiments, the sample cover is a substantially flat lid hingedto the inboard edges of the input queue 628 and the output queue 640.The sample cover may be disposed in either an open or a closed position.When the sample cover is closed, the instrument operates normally andusers may not access the sample presentation unit 110. When the samplecover is open, the instrument may continue to process assays, but doesnot transfer any sample holders 616 into or out of either queue. In someembodiments, the sample cover extends across the entirety of the inputqueue 628 and the output queue 640. In other embodiments, a fixed topmay cover parts of the input queue 628 and the output queue 640 close tothe transfer position 642, and the sample cover in the open positionreveals only a limited portion of the queues.

The mechanized latch may include an actuator mounted to the sample base602, one or more latching hooks mounted to the outboard support rails,and a linkage connecting the actuator to the latching hooks. Theactuator may be any of a number of linear or rotary actuators, includingas solenoids, linear motors, stepping motors, or pneumatic actuators.The latching hooks may align with catches incorporated into the samplecover when the sample cover is in the closed position. The purpose ofthe mechanized latch is to prevent the user from accessing to the samplepresentation unit 110 while the sample holders 616 are in motion. Inoperation, the user requests access by activating a control switch. Thecontrol switch may be implemented utilizing a user interface displayedon a system monitor. The system may respond by completing any sampleholder transfers in progress, reversing the sample pusher to provideroom to add new sample holders 616, cutting power to mechanisms in theinput queue 628, and releasing the mechanized latch. The user may thenopen the sample cover in order to load, unload, or reorganize sampleholders 616. Operations may resume on closing the sample cover.

In an alternative embodiment, the input queue, the output queue or boththe input and output queues may support sample holders in a radial orcircular arrangement. An example of such a circular arrangement is aturntable. In another embodiment a single radial or circular queue mayserve as a combined input and output queue, storing both samples thathave been accessed by the system and unaccessed samples.

In some embodiments, a feature has been added to support the use ofsecure covered tubes during pipetting. These tubes can have a valveassemblies that serve to protect sample tube contents, which aretypically pushed open by the pipette tip during a pipetting operation.These tubes may also have a cap, below which can be a circumferentialridge that is affixed to the exterior wall of the tube. There can be atube stabilizer that inserts into the gap between the cap and thecircumferential ridge to hold covered tubes in place during pipetting.Yet in some embodiments, a sample tube may have a piercable cover orfilm to protect sample tube contents. In such an arrangement, the samplepresentation unit 110 may utilize a pipette tip as a dedicated piercingtool to penetrate through the piercable cover or film to facilitateaccessing the sample contained in the sample tube.

Embodiments of the invention can also include sample holder sensors inthe output queue 640 and the input queue 628. The sensors may include avision system, a barcode reader, etc. Such sensors may also verify thatthe sample holder is properly oriented. The sample presentation unit mayalso include features that support the use of sample tubes withclosures. Such features include sensors that detect the presence ofsample tube caps, and devices for removal or piercing of sample tubecaps in order to provide access to sample tube contents by a samplepipettor. In some embodiments, the sample presentation unit includesfeatures that enhance the stability of sample tube contents. Sample tubetemperature can be controlled by incorporating one or more temperaturecontrolled zones, which may be set to different temperatures. The samplepresentation unit may also include devices, such as infrared sensors,for determining the temperature of the sample tubes held therein. Thesample presentation unit can also include devices for mixing thecontents of sample tubes, such as rocking mechanisms.

C. Sample Pipettor and Pipette Pumps

FIG. 3( a) shows a perspective view of a gantry with a pipette pumpassembly

FIG. 3( b) shows another perspective view of the pipette pump in greaterdetail.

FIG. 3( c) shows details of a compliant coupling used in pipette pumps

A pipette pump or pipettor can be used to transfer liquids from onelocation to another throughout the system. A sample pipettor maytransfer liquids that include patient samples stored in sample tubes,which may include serum, plasma, whole blood, urine, feces,cerebrospinal fluid, saliva, tissue suspensions, and wound secretions.Transferred liquids may also include liquid reagents. Such sample tubesmay be supplied by a user via placement in the SPU 110 described above.Alternatively, sample tubes may be directed to a sample pipettor by alaboratory automation system 80 or by both the SPU and a laboratoryautomation system. The sample pipettor can also interact with thereaction vessel plug 222 and piercer 262 (which are described in furtherdetail below).

Pipettors can also include obstruction detectors (not shown) fordetection of clots in samples and other obstructions. Obstructiondetectors can use a pressure sensor that monitors the pressure profilewithin the pipettor during pipetting events. Certain pressure profilesmay be associated with specific pipettor conditions, includingobstructions and the presence of items attached to the pipettor. Itemsthat may be attached to pipettors include pipette tips, reaction vesselplugs 222, and sealed reaction vessels. Obstruction detectors can alsodetect if a filter is present in a pipette tip, if pipette tips havemolding defects.

Pipettors can also have sensing circuits, such as liquid level sensorcircuits, that can be used to detect contact with a liquid surface.Liquid level sensors can also be used to determine available samplevolume when used in conjunction with encoder information from theelevator motor 730. They can also be used to determine if there issufficient sample volume to perform a test, and one can be used toverify that the correct sample volume was removed from the tube.

In order to reduce contamination, such pipette pumps typically usedisposable pipette tips to contact fluids. A pipette mandrel 728 may actas the point for the attachment of disposable pipette tips to thepipettor. Attachment can be held in place actively by a gripper or heldin place passively by friction between the inner surface of the pipettetip and the outer surface of the pipettor mandrel. The pipette mandrel728 also allows pipette pump assemblies to attach to and subsequentlytransport other consumables that have appropriate interfaces, such as areaction vessel plug or a film piercer, between different locations onthe system. The sensing circuit noted above and described in greaterdetail below can be used to detect the presence of disposable pipettetips, and other consumables that have appropriate interfaces, on thepipettor mandrel. Alternatively, pipette pumps with fixed fluid transferprobes may be used for fluid handling, in conjunction with probe washingmechanisms.

A pipette pump according to an embodiment of the invention may bespecifically constructed to accurately aspirate and dispense fluidswithin a defined range of volumes. Different pipette pumps may be ofsubstantially identical design, with specific components havingdifferent dimensions in order to accurately aspirate and dispense withindifferent volume ranges. In one embodiment, a millitip pipette pump orpipettor can be constructed to accurately aspirate and dispense fluidvolumes ranging from about 50 μL to about 1,200 μL (1.2 mL), and amicrotip pipette pump or pipettor can be constructed to accuratelyaspirate and dispense fluid volumes ranging from about 5 μL to about 200μL. In some embodiments, the system may utilize dual resolution pipettepumps, which are capable of accurately aspirating and dispensing acrossa wide range of volumes, in place of one or more conventional pipettepumps. In an alternative embodiment, liquids may be transferred using apipette pump with a fixed probe or fixed tip, in combination with a washstation for removal of residual liquids following transfer.

One example of a pipette pump assembly is the sample pipettor 700 shownin FIG. 3( a). Reference is also made to certain components in FIGS. 4(a)-4(f). The sample pipettor 700 can be used to transfer aliquots ofsamples from sample tubes to assay cartridges 200. The sample pipettor700 may also serve to transfer fluids from well to well within the assaycartridge 200, add reagents to a sample tube prior to transferring analiquot from a sample tube to an assay cartridge, mix fluids within theassay cartridge 200 (or tubes), puncture holes through a barrier film205 using a piercer 262, and dispose of a piercer 262. The samplepipettor 700 can be located within the system so that it can accesssamples in the sample presentation unit 110 at the aspiration position632 (see FIG. 2( a)) and can reach assay cartridges in the cartridgeloading unit at the sample dispense position. In some embodiments, thesample pipettor 700 can access a waste chute to facilitate safe disposalof solid waste, including but not restricted to a piercer 262.

The sample pipettor 700 may include a sample gantry 718, a pipettorcarriage 712 that supports a millitip pipettor 704, and a liquid sensor702. The liquid sensor 702 may be capacitance based, and may detect bothproximity and contact with liquids and solids that are conductive. Insome embodiments, the sample gantry 718 includes a pipettor carriage 712that carries a sample elevator 710 and is disposed to reach sample tubesand the cartridge loading lane. The sample elevator 710 raises andlowers the millitip pipettor 704 as required for pipetting, mixing,resuspension, and millitip transfer. Alternatively, the sample gantry718 may be any suitable structure capable of reaching the sample tubeand the reaction well such as a rotary transport, a guided tracktransport, an XYZ Cartesian transport, or an articulated arm. A liquidsensor 702 may be incorporated into the sample gantry 718, connecting tothe millitip pipettor 704 and to extensions thereof. Such extensionsinclude disposable pipette tips, reaction vessel plugs, and filmpiercers, which may be constructed of conductive materials. The samplegantry 718 positions the millitip pipettor 704 adjacent each operativelocation, the sample elevator 710 raises and lowers the millitippipettor 704, and the millitip pipettor 704 aspirates, dispenses, orejects the millitip.

An embodiment of the pipettor carriage 712 is shown in FIG. 3( b). Thepipettor carriage can include a pipettor track 715, and a pipettor drive714. The pipettor track 715 can be a section of linear guide railattached to the sample gantry 718 in the direction of travel of thepipettor carriage 712. The pipettor carriage 712 supports the sampleelevator 710 and moves along the pipettor track 715 in response tooperation of the pipettor drive 714. The pipettor track 715 connects tothe pipettor carriage 712 through complementary bearings. In someembodiments, the bearings are caged ball bearing blocks or caged rollerbearing blocks. Although shown with a single pipettor carriage 712 insome embodiments, the sample gantry 718, can support multiple pipettorcarriages, which may in turn carry pipettors with different volumeranges.

Referring to FIGS. 3( a) and 3(b), the pipettor drive 714 may move thepipettor carriage 712 along the pipettor track 715 by any of a number ofdrive methods. Exemplary drive methods include a lead screw and nut, alinear motor, or a pneumatic actuator. In an embodiment shown in FIG. 3(a), the instrument uses a motor attached to the sample gantry 718 nearone terminus of the pipettor track, the motor being coupled to a drivepulley. An idler pulley may be attached to the sample gantry 718 nearthe opposite terminus of the pipettor track 715, by an attachment thatallows adjustment of the separation distance between idler pulley anddrive pulley. A timing belt substantially parallel to the pipettor trackmay connect the drive pulley to the idler pulley couple to the pipettorcarriage 712. The tension of this timing belt may be altered byadjusting the separation between the idler pulley and the drive pulley.Rotation of the motor drives the timing belt and moves the pipettorcarriage 712 along the pipettor track 715.

The sample elevator 710 can be a linear transport that includes anelevator track 708, an elevator carriage 706, and an elevator drive 720.The elevator track 708 can be a section of linear guide rail affixed tothe sample elevator 710 in the direction of travel of the elevatorcarriage 706. The sample elevator 710 can move in a vertical directionin order to move the millitip pipettor 704 into position to accesssample tubes; the elevator track 708 is similarly disposed.

In one embodiment of the invention, the elevator carriage 706 supportsthe millitip pipettor 704, and moves along the elevator track 708 inresponse to operation of the elevator drive 720. The elevator drive 720may move the elevator carriage 706 along the elevator track 708 by anyof a number of drive methods. Exemplary methods include the use of alead screw and nut, a linear motor, or a pneumatic actuator. In someembodiments, the instrument uses a motor attached to the sample elevator710 near one end of the elevator track 708 and coupled to a drivepulley. An idler pulley 734 may be attached to the sample elevator 710near the opposite end of the elevator track 708, by an attachment thatallows adjustment of the separation distance between idler pulley 734and drive pulley. A timing belt 732 substantially parallel to theelevator track 708 runs from the drive pulley to the idler pulley 734and couples to the elevator carriage 706. The tension of this timingbelt 732 may be altered by adjusting the separation between the idlerpulley 732 and the drive pulley. Rotation of the motor 730 drives thetiming belt 732 and moves the elevator carriage 706 along the elevatortrack 708, resulting in vertical movement of the pipettor. The elevatormotor 730 drives the mandrel 728 into the opening of the disposable tip,forming an air tight seal. The tip is held in place by friction, anddetachment may be passive or active.

The sample pipettor 700 may include additional features that supportsample handling functions. The sample pipettor 700 can include areusable film piercing device, configured to pierce the protective filmthat covers a portion of the assay cartridge 200 in one or morelocations in order to provide access to contents. In some embodiments,the sample pipettor 700 includes mixing devices, such as mixing paddlesor ultrasonic probes, that can serve to mix the contents of sample tubesor the assay cartridge 200. The sample pipettor may also include areasfor the storage of reagent bottles.

As shown in FIG. 3( b), the millitip pipettor 704 may include a linearstep motor 722 that is connected to linear actuator 723, which is inturn coupled to a piston 726. The piston 726 lies partially within abarrel 727 that serves as a pressure chamber. A seal lies between thepiston 726 and an inner wall of the barrel 727. The barrel 727 can becooperatively configured to allow movement of the piston within thebarrel. The pipettor 704 may also comprise a mandrel 728 that is influid connection with the barrel 727. Movement of the piston 726 via thelinear step motor 722 generates pressure changes within the barrel 727.These pressure changes are communicated to the mandrel 728 andsubsequently to a pipette tip affixed to the mandrel 728, resulting inthe uptake of fluids into the pipette tip or the dispensing of fluidspreviously held therein. After use the pipette tip can be removed fromthe mandrel 728 by a pneumatically pressurized ejector, which appliespressure to an upper surface of the pipette tip. Alternatively, astripper plate that is driven by the elevator motor 730 may be used toremove the pipette tip. A pipette tip held in place by a gripper may beremoved from the mandrel by releasing the gripping device. The force ofpipette tip ejection can be controllable; for example, the pressureapplied to a mounted pipette tip by an ejector or stripper plate may bevaried, This advantageously permits both slow tip ejections thatminimize the potential for droplet formation and subsequentcontamination and rapid tip ejections that facilitate throughput.

In some embodiments, the connection between the linear step motor 722and the piston 726 incorporates a compliant coupling 724 that connectsthese features. The compliant coupling 724 advantageously simplifiesreplacement of the linear step motor 722, piston 726, housing, and othercomponents of a pipettor, permitting mechanical coupling of the driveand fluid handling components of the device without the need for precisealignment and build tolerances.

FIG. 3( c) shows one embodiment of the compliant coupling 724, where thecompliant coupling 724 can deform slightly along the axis of the linearstep motor 722 and the piston 726 and restricts movement lateral to thisaxis. The compliant coupling 724 may have an upper plate 736 (which isan example of a first connecting feature) and a lower plate 740 (whichis an example of a second connecting feature), these plates beingseparated by a gap, and connectable by an intermediate member 725. Theupper plate 736 can be affixed to the linear step motor 722. In someembodiments, the lower plate 740 has a channel, and the upper portion ofthe piston 726 narrows to pass through this channel, and then flares toa diameter greater than the width of the channel once within the gap.The lower plate 740 is at least partially disposed around the piston726. Compliance is provided by a spring mechanism 738 (or other type ofcompressible member) that lies between the flared portion 726A of thepiston 726 and the lower plate 740 of the compliant coupling 724.Compliance may also be provided by a spring mechanism 738 that islocated outside of this interface and on periphery of the coupling. Inan alternative embodiment, compliance is provided by an elastomericpolymer rather than a spring mechanism 738. This compliance provides theforce desired for a firm connection between the linear step motor 722and the piston 726, which is desirable for accurate fluid dispensing,while reducing the need to build these components to tight tolerances.Additionally, this compliance simplifies replacement of the linear stepmotor 722 or the piston 726 as it reduces the need for careful alignmentof these components. Use of the compliant coupling 724 may not berestricted to the sample millitip pipettor 704, but may be used onpipetting mechanisms throughout the system, or even in systems that aredifferent than the systems described herein.

The millitip pipettor 704 in the sample pipettor 700 may use a millitipdisposable pipette tip associated with each assay cartridge 200 totransfer sample from the sample tube to the assay cartridge reactionwell 202. This advantageously reduces the possibility of contamination,as a different millitip is used in each sample processing instance. Theuse of the relatively large volume millitip allows transfer of a largesample volume. In some embodiments, the sample pipettor 700 picks up themillitip carried within an assay cartridge, transfers the sample aliquotto the reaction well 202 of that assay cartridge, mixes the sample withother materials present in the reaction well 202, and then returns themillitip to a storage position of the assay cartridge 200.

In some embodiments, a pipettor used on the system may use a sensingcircuit, such as a liquid sensor 702, to detect contact with liquidduring pipetting operations. The liquid may be sample held within asample tube or liquid reagents held within an assay cartridge 200 or areagent pack. This detection may be combined with information related tothe position of the pipettor to determine the height of the liquid. Theliquid sensor 702 may incorporate a capacitance-based circuit. Liquidsensing may take place via a conductive pipette tip, such as a millitipor microtip, held on the mandrel 728 of the pipettor. In operation, thepipette tip may be submerged slightly below the liquid surface in orderto limit contamination of the exterior. In some embodiments, thepipettor descends during aspiration to maintain the pipette tip at arelatively constant depth below the sample surface. A sensing apparatusis described in further detail below.

D. Assay Cartridge

Assay cartridges can be one-time use consumables, or may be reusable.There can be many different assay cartridge embodiments. In oneembodiment, the assay cartridge comprises an elongated body comprising adistal end and a proximal end, and a plurality of compartments arrangedlinearly between the distal end and the proximal end, wherein at leastone of the compartments is a reaction well. The reaction well comprisesfirst and second sidewalls, and first and second endwalls, and a wellfloor joining at least the first and second endwalls. The first endwallcomprises a plurality of bends, which can form a faceted shape.

The various compartments in the assay cartridge can include DNA reagentcompartments for storing reagents for DNA extraction from a sample, orRNA reagent compartments for storing reagents for RNA extraction from asample.

In a specific embodiment, the assay cartridge comprises a reaction wellincluding a first sidewall, a second sidewall, a first endwall, a secondendwall, and a well floor arranged to receive a reaction mixture. Thefirst sidewall, the second sidewall, the first endwall and the secondendwall form an open end. The first endwall includes a first segment anda second segment. The first and second segment are joined by a bend, andat least one of the first segment and second segment is tapered so thatthe cross section of the reaction well decreases closer to the wellfloor.

FIG. 4( a)-1 shows one embodiment of an assay cartridge 200. The assaycartridge 200 comprises an elongated body 201 formed to include multiplecompartments, which may hold fluids (e.g., reagents) and devices (e.g.,millitips) needed to perform various analyses. Examples of compartmentsmay include one or more reaction wells 202, one or more millitip holders203, one or more large reagent wells 204, one or more medium reagentwells 208, and one or more small reagent wells 209. In some embodiments,the assay cartridge 200 can be in the form of a monolithic body, and maybe formed of plastic (or any other suitable material). In some cases, aplastic injection molding process can be used to form the assaycartridge 200. Alternatively, the assay cartridge 200 may be constructedby fitting individual components into a rigid framework.

Each assay cartridge may also include a containment region 212, a cover(e.g., a barrier film 205) which is disposed around variouscompartments, features to facilitate handling and automation (e.g., adetection feature 210), selected reagents, labeling, and removablecomponents that can be used during processing. The assay cartridge 200can have a proximal end 230 and a distal end 232 at opposite ends of theelongated body 201. The orientation of the compartments defines the topand bottom portions of the assay cartridge 200. In some embodiments,compartments can be open at the top and closed on the bottom and sides.

As shown in FIG. 4( a)-1, compartments within an assay cartridge canalign in a single file. This linear layout allows simple linear motionto align each compartment of the assay cartridge with operativelocations in linear processing lanes. Alternatively, assay cartridgesmay take other shapes such as an arc, a multi-row grid, or a circle,among others. The choice of shape for an assay cartridge can depend onthe overall system design, such as on the number and sequence ofoperative locations that need access to the individual compartmentswithin an assay cartridge. The described linear assay cartridge designis advantageous, because it supports compact storage of assaycartridges, compact layout of processing lanes that operate on the assaycartridges, and easy user handling of multiple assay cartridges. It isalso relatively simple to manufacture.

In some embodiments, the top ends of compartments within an assaycartridge form openings that align at a common height. In some cases,compartment bottom ends generally do not align because compartmentsdiffer in depth and because the compartment bottoms may have differentshapes. The common height facilitates use of shared closures to reducecontamination risk at lower cost. It also reduces the effect of assaycartridge tolerance stackup on system alignment, since the system maysupport assay cartridges during processing from a controlled surfaceclose to the assay cartridge top.

In some embodiments, assay cartridges have a skirted containment region212 surrounding the openings of each compartment. The containment region212 can be defined by a first longitudinal wall 206, a secondlongitudinal wall 207 substantially parallel to the first longitudinalwall 206, a first transverse wall 213, and a second transverse wall 214.Walls 206, 207, 213, and 214 may be referred to as “skirting walls” insome embodiments of the invention. In some embodiments, the assaycartridge 200 may have multiple skirting walls that serve to containassay well contents that might otherwise be sources of contamination.The first and second transverse walls 213, 214 may be substantiallyperpendicular to the first and second longitudinal walls 206, 207, suchthat the containment region 212 is defined by a rectangle in thisembodiment. The longitudinal walls 206, 207, and the transverse walls213, 214 can extend above the upper openings of the variouscompartments. The transverse walls 213, 214 help to contain any drips orspills that may occur during assay cartridge processing. The transversewalls 213, 214 surround the openings of the compartments to create anextended cavity open at the top and contiguous with the interior of oneor more compartments. The containment region 212 may further be definedby a horizontal web 228, which may connect between the compartmentopenings and the transverse walls 213, 214. The horizontal web 228 formsa floor for the containment region 212 and a support for the compartmentwalls 206, 207, 213, 214. The bottom surface of the horizontal web 228can be a controlled surface that the system uses to support each assaycartridge during processing.

Compartments within the assay cartridge can perform a variety offunctions. For example, component storage compartments can storeremovable components such as millitips. Reagent wells can storereagents. A reaction well can provide a reaction site. In addition, somecompartments may perform more than one function. For example, reagentwells initially contain reagents used in processing the assay cartridge,and some reagent wells may later hold wastes produced during assaycartridge processing. Used compartments can hold discarded components(microtips, piercer, and vessel cover) in addition to discarded fluids.

Generally, compartments in some embodiments lack common walls to preventthe creeping of liquids between compartments. This has the benefit ofreducing the possibility of contamination between compartments. Lack ofcommon wells also supports leak testing of reagent wells during assaycartridge manufacture. In some embodiments, the external profile of eachcompartment closely tracks the cavity internal profile. That is, thewalls can be of relatively constant thickness and can be thin withrespect to the size of the compartment. This has the benefit of reducingthe amount of material used and hence reduces the manufacturing cost ofthe assay cartridge. An additional benefit of thin compartment walls andconstant thickness is more efficient and consistent heat transfer, whichcan be desirable for temperature control. Relatively constantcross-sections also contribute to more consistent parts with injectionmolded assay cartridges. The walls that define each compartment mayextend as rims above the horizontal web both to prevent the incursion offluids dripped or spilled in the containment region, and to act asenergy directors to attach closures to the compartments. These rims mayalso support leak testing of reagent wells during assay cartridgemanufacturing. The walls of the compartments may extend slightly abovethe horizontal web to act as energy directors for attachment ofclosures. They can also act as heat sealing contacts.

In some embodiment, a vertical web 226 disposed generally along thelongitudinal axis of the assay cartridge may connect the compartmentwalls. The vertical web 226 may extend beyond the compartments to atleast partially define the external profile of the assay cartridge 200.This has benefits of conferring rigidity to the assay cartridge, ofcontrolling the fit of assay cartridges in the instrument loading area,and of providing space for labels and other indicia. An additionalbenefit of the vertical web 226 is to assist in the flow of plasticthrough the mold during the injection molding process. The vertical web226 may also provide a location for keying features used to designatecartridge type and prevent insertion into the wrong lane of thecartridge loading unit. It can also be a support for human and machinereadable information such as machine readable one and two dimensionalbarcodes. The assay cartridge 200 may also include other verticalextensions that provide lateral stability and allow it to be freestanding.

Component storage compartments within assay cartridges may hold discretecomponents used in the extraction and purification process or in theamplification process. In some embodiments, one compartment can be amillitip holder 203, which supports a millitip pipette tip 220. Othercompartments can include reaction vessel component holders 219, whichcan hold components of a reaction vessel. Components of a reactionvessel may comprise a vessel base 246 and a vessel plug 222, which canfit within the vessel base 246.

In some embodiments, each storage compartment supports its associateddiscrete component at a common operating height. The operating height isthe height at which the discrete component interacts with instrumenttools. In some embodiments, one or more walls 213 extend between atleast some of the storage compartments and connect to the longitudinalwalls 206, 207 to segregate at least some of the discrete components.

Reagent wells within assay cartridges may be of several types. Amongthese may be small reagent wells 209 that hold small volumes ofreagents, medium reagent wells 208 to hold solid phase microparticles orto contain intermediate volumes of reagents, and large reagent wells 204that may hold wash fluids, buffers, other reagents, or sample. Reagentsstored in reagent wells may be in the form of liquids or particlessuspended in liquid. In some embodiments, reagents stored in reagentwells are in the form of lyophilized solids, lyophilized pellets, or dryfilms adhered to the interior walls of the reagent wells. Some reagentwells may be empty. A barrier film 205 can close the tops of the reagentwells.

Small reagent wells 209 may hold materials used in small amounts. Smallreagent wells 209 may be cylindrical with conically tapered bottoms.This shape minimizes dead volume and allows a pipettor to collect all,or nearly all, of the contained reagent. In some embodiments, each assaycartridge 200 has one small reagent well 209 with a fill volume of about200 microliters (or more) with a headspace allowance of about 7.6 mm (ormore). Small reagent wells may also be rectangular with pyramidalbottoms to (a) direct liquid volumes to the bottom of the well and (b)improve conductive heat transfer when a heating element is applied tothe external walls. Small reagent wells may also have a rectangularcross-section in some embodiments of the invention. Bottoms may be havea central deepest point, and may be rounded, conical, pyramidal. Abenefit of well with a rectangular cross-section is that flat contactareas provide for improved thermal contact/temperature control.

Medium reagent wells 204 hold reagents needed in relatively smallvolumes or reagents that may need mixing during use. For example, mediumreagent wells 204 may hold the solid phase microparticles. In someembodiments, the system stores solid phase microparticles in suspension,but dry storage may extend shelf-life. In either case, solid phasemicroparticles may require mixing before use either to resuspendmicroparticles that settle in storage or to disperse a rehydratedsuspension. Other medium reagent wells may hold reagents not requiringmixing or another mixture, such as a mixture of sample and a diluent,which the system may form preparatory to transfer into the reactionwell. In some embodiments, each assay cartridge 200 has two mediumreagent wells, each with a fill volume of about 350 microliters (ormore) with a headspace allowance of about 7.6 mm (or more). Mediumreagent wells may also have a rectangular cross-section in someembodiments of the invention. Bottoms may be have a central deepestpoint, and may be rounded, conical, or pyramidal. A benefit of well witha rectangular cross-section is that flat contact areas provide forimproved thermal contact/temperature control.

Medium reagent wells 208 may have a rectangular cross-section, withpyramidal bottoms. This conformation advantageously directs liquidvolumes to the bottom of the well and improves conductive heat transferwhen a heating element is applied to the external walls. In otherembodiments, the medium reagent wells can be cylindrical with roundedbottoms, and in some cases with hemispherical bottoms. In someembodiments, the system mixes medium reagent well contents using tipmixing. Tip mixing can include one or more cycles of aspiration andredispense of the contents. For example, the tip could be a millitip andaspiration and redispense of the contents may be performed using themillitip. Tip mixing agitates the contents so that different elements ofthe fluid interact on a small scale. The pyramidal or hemisphericalbottoms of the medium reagent wells 208 support agitation and limitedrotation of the redispensed contents with a minimum of uninvolvedvolume. The redispense process uses the kinetic energy of theredispensed fluid to impel fluid agitation. The medium reagent well 208has a diameter that is a relatively large fraction of the width of theassay cartridge to reduce the effects of capillary forces on mixing. Themedium reagent well 208 has a depth greater than its diameter to bettercontain any splashing. In some embodiments, the depth of the mediumreagent well is at least twice its diameter; the diameter may be atleast about 1 mm (e.g., between about 1 and 10 mm) and in some cases atleast about 5 mm.

The system may use any of a number of other methods to mix reagent wellcontents. For example, the system may accelerate the assay cartridge 200in one or more dimensions to agitate contents, or it may use a pipettetip or other device disposed in the fluid as a mixing tool. Other mixingmethods may include magnetic mixing, ultrasound, and rotating paddles orsimilar devices that are inserted into the wells.

Large reagent wells 204 may hold wash fluids, buffers, other reagents,wastes, or sample. Generally the system uses large reagent wells 204 toaccommodate relatively large volumes of reagents or to accommodatereagents that are sufficiently homogeneous as not to require mixing.Even so, the system may mix materials in large reagent wells by, forexample, the tip mixing process described above. Large reagent wells 204can taper to minimize dead volume and hence allow a pipettor to collectall, or nearly all, of the contained reagent. In some embodiments, thetaper is at least a two part taper to allow a relatively large volumepipette tip with a shallow taper to reach the bottom of the largereagent well 204. The taper has the added benefit of acting as a draftthat eases ejection of the assay cartridge 200 during fabrication. Insome embodiments, assay cartridges have seven large reagent wells, eachwith a fill volume of about 2000 microliters with a headspace allowanceof about 7.6 mm. Large reagent wells may also have a rectangularcross-section in some embodiments of the invention. Bottoms may be havea central deepest point, and may be rounded, conical, pyramidal. Abenefit of well with a rectangular cross-section is that flat contactareas provide for improved thermal contact/temperature control. The flatexterior walls of the large reagent wells may be used to support labels,barcodes, and other indicia.

A barrier film 205 may seal the reagent wells individually to preservethe reagents and to prevent reagent cross-contamination. In someembodiments, a single barrier film 205 may cover all reagent wells. Inanother embodiment, the reagent wells of the assay cartridge 200 mayhave individual seals. The barrier film 205 may be a multilayercomposite of polymer and foils, and can include metallic foils. In someembodiments, the barrier film 205 includes at least one foil componentthat has both a low piercing force and sufficient stiffness to maintainan opening in the barrier film 205 once the piercing device is removed.Additionally, the barrier film 205 may be constructed such that nofragments of the foil component are released from the barrier film uponpiercing. A suitable material for the barrier film may be Part No.AB-00559 supplied by Thermo Scientific, Inc. of Epsom, UK. The barrierfilm 205 can be a continuous piece spanning all of the reagent wells. Inoperation, a pipette tip pierces the barrier film to access reagent wellcontents. The manufacturing process may pre-score the barrier film sothat any tearing upon piercing occurs in predictable locations. In someembodiments, the manufacturing process laser welds the barrier film tothe rims of each reagent well. Alternatively, the manufacturing processmay use other attachment methods to fix the barrier film to the reagentwells. Other suitable processes may include heat sealing, ultrasonicwelding, induction welding, or adhesive bonding.

FIG. 4( a)-2 shows a top perspective view of another assay cartridgeaccording to another embodiment of the invention. The assay cartridge200 shown in FIG. 4( a)-2 is similar to the assay cartridge 200 in FIG.4( a)-1, except that the side walls of the medium reagent wells 208′ aresubstantially flat and the openings of the reagent wells 208′ aresubstantially parallelepipeds (e.g., squares). The side walls of thereagent wells 208′ are substantially curved and the openings of thereagent wells 208 are substantially round in the assay cartridge 200 inFIG. 4( a)-2. The flat side walls of the reagent wells 208′ canadvantageously be in better thermal contact with a heater compared tothe curved side walls of the reagent wells 208 thereby providing betterheat transfer to reagents in the reagent wells 208′.

FIG. 4( b) shows a side cross-section view and a top plan view of areaction well 202 in the assay cartridge 200.

Referring to both FIGS. 4( a) and 4(b), the assay cartridge 200 includesat least one reaction well 202 that contains reaction mixtures duringthe extraction and purification process. While the system operates onother assay cartridge compartments primarily from the top, the reactionwell 202 can also interact with tools such as magnets and heatersthrough its sides and edges. For this reason, in one embodiment, thereaction well 202 can reside near one end (the proximal end) of theassay cartridge 200. This end positioning advantageously allows tooloperation by moving the assay cartridge 200 to place the reaction well202 close to the tools. The end positioning has the further benefit ofreducing the possibility of contamination by avoiding transporting thereaction well under an active pipette tip, except while pipetting to orfrom the reaction well 202. Placement of reaction well at one end alsoreduces risk of contamination entering the reaction vessel during mixingactivities.

The reaction well 202 has a faceted shape (which may be formed byrectangular segments) designed to contain a relatively large reactionvolume, to permit effective mixing of its contents, to permit aspirationwith minimal dead volume, to assure good thermal contact with externalheaters, and to interact with external magnets at either high or lowfill volumes. The reaction well 202 can have a capacity of about 4500microliters with a headspace allowance of about 7.6 mm. This relativelylarge capacity supports the processing of sample volumes in themilliliter range. The ability to process large sample volumes reducessampling error and improves detection of rare sequences that may bepresent at only a few copies per milliliter of sample. In otherembodiments, the reaction well can have a gradual transition designinstead of a faceted shape. In some embodiments, the combination ofreaction well volume and its faceted shape permits both the processingof large sample volumes and the recovery of small volumes, allowing itto be used for sample concentration and hence detection of raresequences.

As shown in FIG. 4( c)-1, the reaction well 202 can have a generallyrectangular cross-section (in the plane of the horizontal web) with thelong axis of the rectangle aligned with the long axis of the assaycartridge 202. The reaction well 202 can be at least wide enough toaccommodate the millitip pipette tip 220. The reaction well 202 taperswith depth both from its sidewalls (generally parallel to the assaycartridge axis), which may include first and second sidewalls 202(c),202(d), and from its endwalls (generally perpendicular to the assaycartridge axis), which may include first and second endwalls 202(a),202(b). The first and second sidewalls 202(c), 202(d) have a dual taperwith a shallow draft (closer to vertical) for most of the height and asteeper draft (closer to horizontal) near the reaction well floor 240(shown in FIG. 4( b)). The first and second sidewalls 202(c), 202(d)converge in the steeper draft portion to narrow the reaction well nearits floor 240.

In the longitudinal section along the assay cartridge axis, the reactionwell 202 can be asymmetric, with a deepest portion aligned relativelyclose to that endwall 202(b) distal from the assay cartridge proximalend 230 (see FIG. 4( a)-1). As shown in FIG. 4( b), this deepest portionfits a millitip pipette tip 220 so that the millitip 220 can reach thedeepest portion without touching the sidewalls when the millitip is inan aspirate position 236 (which can correspond to a second location insome cases). The pipette tip 220 may include a coupling taper 220(a) atone end and a pipetting orifice 220(b) at the other end. Thelongitudinal section profile of the reaction well can be polygonal, andthe bottom can rise in a piecewise linear fashion to join the endwallproximal to the proximal end 230 of the assay cartridge 200. Eachsuccessive segment (beginning at reaction well floor 240 andrespectively bounded by first bend 202(a)-1, by second bend 202(a)-2,and by third bend 202(a)-3), aligns closer to the vertical. The anglesof these successive segments may be obtuse relative to the verticalaxis. In one embodiment the angle of the internal surface of the firstsegment (extending from reaction well floor 240 to bend 202(a)-1) rangesfrom 100° to 120° relative to the vertical axis, the angle of theinternal surface of the second segment ranges from 135° to 155° relativeto the vertical axis, and the angle of the internal surface of the thirdsegment ranges from 150° to 170° relative to the vertical axis. Thesegment extending beyond third bend 202(a)-3 may be approximatelyparallel to the vertical axis. In another embodiment, the angle of theinternal surface of the first segment is about 110° relative to thevertical axis, the angle of the second segment is about 145° relative tothe vertical axis, and the angle of the third segment is about 160°relative to the vertical axis. In some embodiments, the reaction welllongitudinal section profile along the assay cartridge axis planeincludes four linear segments (defined by the first, second, and thirdbends 202(a)-1, 202(a)-2, 202(a)-3) between the deepest point and thereaction well top at the proximal end. Two linear segments can connectthe deepest point and the reaction well top at the distal end. Thepreviously described bends may be rounded transitions that linksuccessive linear segments. However, the transitions that linksuccessive segments may be angular where it is desirable to confinestanding liquids.

In embodiments of the invention, a first segment (above bend 202(a)-3)is closer to the open end of the reaction well and has a first taper,the second segment (e.g., below bend 202(a)-2) is farther from the openend and has a second taper. The second taper can be being greater thanthe first taper so as to decrease the cross-section of the reaction wellat a greater rate.

The proximal endwall (i.e., the first endwall 202(a)) tapers towards theproximal end of the end of the assay cartridge and towards the bottom ofthe reaction well. As proximal endwall 202(a) approaches reaction wellfloor 240, sidewalls 202(c) and 202(d) converge toward the reaction wellmid-line (or mid-plane). The cross-section of the reaction well candecrease towards the well floor 240. The lower segments of proximalendwall 202(a) and converging sidewalls 202(c) and 202(d) may intersectin a smooth curve. The radius of this curve decreases towards reactionwell floor 240, thereby forming a segment of a frusto-conical surfacedefining culvert 211. The smooth walls of culvert 211 serve to funnelfluid towards reaction well floor 240. The culvert 211 directs fluidadded from above the culvert toward the reaction well midline so as toengulf and induces turbulence to scour any materials localized on thelower proximal endwall. In some embodiments materials localized on thelower proximal endwall include magnetically responsive particles. Theculvert 211 may also enhance mixing of reaction mixtures.

The faceted geometry of the reaction well 202 can permit effectivemixing of reaction well contents using a modified tip mixing protocol.The system can mix by aspirating reaction well contents with themillitip 220 in or near the deepest portion of the reaction well 220.The system then redispenses the aspirated material with the millitip 220nearer the proximal sidewall in a dispense position 234 (which maycorrespond to a first location in some cases), roiling and mixing thefluid. In some embodiments, the system redispenses the aspiratedmaterial with the millitip 220 onto the culvert 211, inducing turbulencewhile roiling and mixing the liquid. Particulates, such asmicroparticles, that have been deposited on the culvert may be suspendedby such mixing. Such mixing actions may be repeated by re-aspirating thedispensed liquid and re-dispensing it. The system can aspirate from thereaction well using either a millitip or a microtip.

Aspirating with the millitip 220 at or near the deepest point minimizesdead volume. The angled floor of the reaction well 202 in this regionprevents formation of a seal between millitip 220 and reaction wellfloor 240 that might otherwise block the millitip during aspiration. Theintersections that define the bends between linear segments of thefacets can serve to segregate volumes of liquid from materials localizedwithin the culvert 211.

The culvert 211 also advantageously amplifies the scouring effect ofadded fluid to wet and resuspend solid phase materials. The narrowingand incurving shape of the culvert directs even small volumes of fluidwith increased velocity to help resuspend magnetic materials previouslypulled to the culvert's lower portion. The proximal first endwall 202(a)may curve outwards proximally in order to give the culvert an ovalcross-section. This acts to contain materials localized against thisportion of the proximal endwall in a defined area along the midline,enhancing the scouring action of added fluid and physically isolatingsuch contained materials from small eluent volumes. This is particularlyadvantageous in the late steps of nucleic acid isolation when a smalleluent volume is desirable.

FIG. 4( c)-2 shows a top plan view of another reaction well according toanother embodiment. In FIGS. 4( c)-1 and 4(c)-2, like numerals designatelike elements. In FIG. 4( c)-2, the culvert 211 is formed by relativelystraight side boundaries, whereas the culvert 211 in FIG. 4( c)-1 hascurved side boundaries.

In some embodiments, the external profile of the reaction well 202closely tracks the cavity internal profile. That is, the walls202(a)-202(d) are of relatively constant thickness and thin with respectto the size of the reaction well 202. In addition to the benefitsdiscussed above, this advantageously improves thermal conduction betweenexternal heaters and reaction well contents. Better thermal conductionreduces the time for reaction well contents to reach desiredtemperatures, decreasing the length of processing and assuring moreuniform conditions within the reaction well. More uniform conditionscontribute to better repeatability in nucleic acid isolation and henceto more precise answers. Alternatively, the reaction well 202 may havewalls of relatively uniform thickness but of reduced thickness inregions of contact with external heaters.

The faceted shape of the reaction well 202 also supports interactionwith external magnets at either high or low fill volumes by providing anextended region for magnetic coupling. The extended region may be afacet of the reaction well 202 forming a segment of the proximal firstendwall. The external surface of an endwall segment can be disposed atan acute angle with respect to the vertical axis of the reaction well.In some embodiments, the acute angle can be between about 20 degrees andabout 70 degrees and in some cases about 35 degrees. This acute angleadvantageously allows juxtaposition of either a relatively large magnetor a smaller magnet proximate the facet. Either size magnet so disposedsets up a magnetic field that collects and pellets magneticallyresponsive microparticles adjacent the interior reaction well firstendwall 202(a) in the culvert 211. A smaller magnet can collect themagnetically responsive microparticles along the culvert surface nearthe bottom of the reaction well 202. A large magnet also collects themagnetically responsive microparticles along the culvert 211 butdistributes them over a larger portion of the interior surface. Thelarger magnet may collect the magnetically responsive microparticlesmore rapidly, and the system can more readily resuspend the distributedpellet. Both of these attributes reduce processing time. The smallermagnet spatially limits the distribution of magnetically responsivemicroparticles so that addition of a small volume of fluid reachesessentially all of the smaller pellet. This is advantageous when thesubsequent processing step adds only a small volume of fluid. This mayoccur, for example, immediately prior to elution of the nucleic acidwhere a minimal elution volume is desirable.

FIG. 4( j) shows a number of side cross-sectional views of alternativereaction well embodiments 202-1, 202-2, 202-3, 202-4, 202-5. Each designhas a different endwall configuration. A millitip 220 is shown with eachreaction well design. Reaction well embodiment 202-1 has a configurationthat is somewhat similar to the reaction well shown in FIG. 4( b).Reaction well embodiments 202-2, 202-3 have fewer angled portions in theendwall leading to the bottom than the reaction well embodiment 202-1.Reaction well embodiments 202-4, 202-5 show embodiments where endwallportions of the reaction wells are curved. Reaction well embodiment202-4 is shorter and has less volume than reaction well 202-5.

The assay cartridge 200 may be made of any suitable material. Forexample, the assay cartridge 200 may comprise of a hydrophobic polymer,such as polypropylene. If this is the case, the interface betweenaqueous buffers and the assay cartridge can have a high angle ofincidence. This high angle of incidence can localize the air/liquidinterface of an appropriate volume of buffer along the line defined byan angular intersection between the culvert facet and an adjacent facet.This volume may be between about 1 microliter and about 100 microliters,and in a preferred embodiment is about 25 microliters. The assaycartridge 200 could alternatively comprise polyethylene, fluoropolymers,polystyrene, silicone, and copolymers thereof, and these and othermaterials could be applied as films or layers over other materials

Assay cartridges may include a removable cartridge cover (not shown) toprotect contents prior to use. Covers may be made of plastic, paper orcardboard that fit on or near the top of a containment wall of the assaycartridge. The cover advantageously reduces the possibility ofcontamination during storage and handling. In some embodiments, the userremoves the cartridge cover at about the time she loads assay cartridgesinto the system. Alternatively, assay cartridge packaging may integratethe cartridge cover such that removal of the assay cartridge from thepackaging also removes the cartridge cover. The cartridge cover mayadhere to the assay cartridge by a snap fit or similar method but insome cases, the cartridge cover forms a “tear-off” strip adhered to thetop of the skirting walls. A flexible barrier material such as paper,Tyvek®, or a polymer film can form the body of the tear-off strip.Adhesion of the tear-off strip to the skirting walls may be by any of avariety of techniques such as adhesive bonding or ultrasonic welding,and is in some cases thermal bonding. In use, a user may simply peel thetear-off strip from the assay cartridge. Optionally, the cartridge covermay include preprinted instructions or other information.

Referring to FIG. 4( d), the assay cartridge 200 includes features tofacilitate handling and automation. These features include surfacescontrolled during manufacture to establish one or more positioningreferences, support tabs 218 to support the assay cartridge 200 duringstorage and to position an assay cartridge during processing, acartridge flange to retain the assay cartridge during withdrawal of apipette tip, a detection feature (see element 210 in FIG. 4( a)-1) todiscriminate adjacent assay cartridges, asymmetric features to preventinverted loading of assay cartridges, keying features to distinguishtypes of assay cartridges, and marking elements to transfer informationrelated to assay cartridges.

Controlled surfaces facilitate assay cartridge position by providingreference locations that the manufacturing process holds to tighttolerances. In some embodiments, one controlled surface is thevertically disposed edge of the vertical web at the distal end of theassay cartridge. The bottom surface of the horizontal web may be acontrolled surface.

FIG. 4( d) shows an end of the assay cartridge 200 with a support tab218, which engages a propelling feature 303 of a cartridge carriage.

In embodiments of the invention, a pair of support tabs 218 may supportthe assay cartridge 200 on the system. Support tabs 218 protrude fromeither end of the assay cartridge, and each support tab 218 includes ahorizontal element and a vertical element. Parallel rails within thesystem (e.g., within a cartridge loading unit) may retain the assaycartridges by providing support for the horizontal elements from below.The vertical elements extend downward from the horizontal elements.Similar spacing of the vertical elements and of the parallel railsaligns the assay cartridges on the parallel rails. In some embodiments,the vertical elements are further from the assay cartridge midpoint thanare the horizontal elements. That is, the horizontal elements extendperipherally from the assay cartridge and terminate in the verticalelements. This has the benefit of preventing a misaligned assaycartridge from falling between the parallel rails.

The support tabs 218 may also position the assay cartridge duringprocessing within the processing lanes. While the system may push orpull assay cartridges from either end, avoidance of tolerance stackupfavors pushing or pulling consistently from a single end. Accordingly,assay cartridges may have a more robust support tab at one end toprovide greater rigidity for this more demanding use. In someembodiments, this more robust support tab integrates a vertical I-beamstructure into the vertical element and connects it to the bottom of themost distal compartment. The support tab 218 on the distal end of theassay cartridge 200 depends from the assay cartridge a small distancedistal to the controlled surface defining a gap. Support tabs 218 on theassay cartridge may also be used to support the assay cartridges whenheld within packaging. The gap between the support tab 218 and thedistal surface of the assay cartridge 200 may also be tapered tofacilitate transfers of the assay cartridge within the system.

The assay cartridge 200 can also include features to retain the assaycartridge during withdrawal of a pipette tip. Such features may be ofparticular benefit when the system removes a pipette tip 220 from areagent well covered by a barrier film 205. Assay cartridge retentionfeatures are also useful when using the piercer to penetrate the sealover the compartments. As discussed above, the barrier film 205 mayinclude components that exert friction on a pipette tip 220 as thesystem withdraws the pipette tip from the reaction well. Withoutfeatures to retain the assay cartridge 200, the pipette tip 220 may liftthe entire assay cartridge 200 from its support, displacing it orcausing splashing and subsequent spills when the assay cartridge 200drops back down to the support. The barrier film 205 may also containbrittle or rigid components, such as foils, that hold the hole in thefilm open after piercing so as to not interfere with subsequentpipetting operations.

In some embodiments, the assay cartridge 200 includes a cartridge flangedisposed on at least one edge of the assay cartridge 200. Such acartridge flange may be an extension of the horizontal web extendingbeyond the skirting wall for at least a portion of the length of theassay cartridge. The cartridge flange may protrude at a height slightlylower than the horizontal web to support closer packing of assaycartridges when disposed side by side. In some embodiments, thecartridge flange extends substantially the entire length of the assaycartridge. The system may also or alternatively use some other feature,such as the top of the skirting wall, to retain the assay cartridge. Thepresence of the cartridge flange also supports manual handling ofmultiple assay cartridges.

Assay cartridges may include detection features 210 to discriminateadjacent assay cartridges when the instrument stores multiple assaycartridges together. The purpose of such detection features 210 is topermit the instrument to sense the presence of loaded assay cartridgeswithin the loading area. For example, the first and second longitudinalwalls 206, 207 can extend around the entirety of the assay cartridge 200above the horizontal web 228. The first and second longitudinal walls206, 207 may determine the separation distance between assay cartridgeswhen disposed side by side such that an external sensor responsive tothe longitudinal wall at one end of the assay cartridges might notreadily distinguish one assay cartridge from another. In someembodiments, the longitudinal wall at the distal end has reduced extentcompared to the distance between longitudinal walls along the assaycartridge sides. The distal end portion of the longitudinal wall mayinclude two or more segments, where one segment is disposed at or nearthe distal end of the assay cartridge and other segments are disposedinward of the distal end. The segments can connect to each other byshort transverse segments of longitudinal wall disposed generallyparallel to the assay cartridge axis. This segmented geometry retainsthe complete containment of the longitudinal wall and allows an externalsensor placed near the distal end to discriminate the segment disposednear the distal end from the rest of the assay cartridge.

Assay cartridges may include asymmetric features to prevent a user frominadvertently loading assay cartridges backwards, i.e. end-for-endreversed. The system may include features in the assay cartridge loadingarea complementary to these asymmetric features but not complementary toa reversed assay cartridge. Thus, the assay cartridges may only fit inone orientation in the loading area. Assay cartridge asymmetric featuresmay be a natural consequence of the distribution of differently sizedand shaped compartments. For example, the millitip pipette tip hassufficient capacity to transfer the content of a reagent well in asingle aspiration, but the millitip diameter can be less than thereagent well diameter in order to reach the reagent well contents. Themillitip can therefore be longer than the depth of the reagent well, andthe compartment within the assay cartridge that supports the millitip isthus deeper than the reagent well. Since each assay cartridge includes asingle millitip pipette tip, and since the millitip may be adjacent thereaction well near the proximal end of the assay cartridge, the assaycartridge may have greater height near its proximal end than near itsdistal end. Alternatively, the vertical web of the assay cartridge mayhave an asymmetrical shape.

Assay cartridges may include keying features 224 to distinguish types ofassay cartridges during user loading of assay cartridges within theloading area. The purpose of this keying is to avoid inadvertentmisloading of different assay cartridge types. The keying prevents assaycartridges of one type from fitting into a portion of the loading areadesignated for a second type. In some embodiments, the keying featuresare rectangular cutouts at the bottom of the vertical web. The positionof the cutouts along the length of the assay cartridge may be unique foreach assay cartridge type.

Assay cartridges may include marking elements to transfer information.Marking may include machine readable information in any of a variety offorms such as a barcodes, dot codes, radio frequency identification tags(RFID) or direct-reading electronic memory. In addition, human readableinformation such as text or illustrations may also be present. In someembodiments, each assay cartridge includes a barcode on the vertical weband text on the vertical web, on the longitudinal walls, and on theremovable cover. The marking may include information about assaycartridge type, manufacturing information, serial numbers, expirationdates, use directions, and similar information.

Assay cartridges can contain at least some reagents used in isolationand purification of nucleic acids. Assay cartridges may also containsome reagents used in amplification and detection. Among the reagentsmay be wash fluids, buffers, diluents, eluents, microparticles, enzymes,cofactors, or other reagents. In some embodiments, the system first usesmaterials from reagent wells nearest the reaction well. When removingwastes, the system first deposits waste material in empty wells closestto the reaction well. This advantageously reduces the possibility ofcontamination, as droplets falling from a pipette tip can only fall intowells that the system has already used.

During processing, assay cartridge compartments contain in-processmaterials. Although most in-process materials reside in the reactionwell, others, such as neat or diluted samples, reconstituted reagents,eluted nucleic acids, wastes, or others, may reside in othercompartments at various times during processing. Among the wastesretained may be liquid wastes such as expended reactants and solidwastes such as expended pipette tips. Placement of the millitip holdernext to the reaction well reduces the chances of contamination of openreagent wells by the millitip, as it is placed in the millitip holderafter processing contents of the reaction well potentially contaminatingdrips fall into bottom of millitip holder on ejection of the millitip.

In some embodiments, the system uses materials from reagent wells in asequence that is roughly based on the position of the reagent wells inthe assay cartridge. The system may limit transfers (other than tipmixing) to a single aspiration from each reagent well in order to avoiduse of material possibly contaminated by an earlier aspiration. Thesystem may first use materials from reagent wells nearest the reactionwell. When removing wastes, the system first deposits waste material inempty wells closest to the reaction well. This sequencing of well usageadvantageously reduces the possibility of contamination. Any dripsfalling from the pipettor can only fall in wells that the system hasalready used.

Prior to loading on the system, assay cartridges may be stored intransport boxes. A transport box retains several assay cartridges incommon orientation, grouped for easy grasping of several at a time toload. In some embodiments, transport boxes include a supporting base,labeling, and a clamshell lid to protect the assay cartridges duringhandling. Storage slots in the supporting base may group assaycartridges as two sets of three to five with a gap in between the sets.Manufacturing processes useful for producing transport boxes include atleast plastics thermoforming and plastics injection molding.

Some embodiments of the invention are also directed to a disposable filmpiercer. As noted above, the assay cartridge 200 has a barrier film 205that lies over and seals the reagent wells 204, 208, 209 prior to use.The millitip 220 pipette tip can be used to penetrate the film. Themillitip 220 can have features incorporated into the tip to equalize theair pressure as it pushes through the barrier film 205. In someinstances, this could cause contamination problems. For example, inprotocols where the millitip first draws patient sample; some residualsample may be retained on the exterior surface of the millitip and inpressure equalizing features. When the film 205 is subsequentlypenetrated by the millitip the initial stretching of the filmpressurizes the interior of the sealed well. This may generate a smallburst of air that exits around the exterior of the millitip on actualpenetration, which may atomize such residual sample. It is possible thatthe patient sample could be spread beyond its intended area. To helpsolve this problem, some embodiments of the invention can use a separatefilm piercer.

FIG. 4( e) shows a perspective view of a film piercer 262 according toan embodiment of the invention. As shown, the film piercer 262 comprisesa linear piercing element 266 comprising a piercing element end 266(a),which is sharp, and a pipette mandrel interface 267. The pipette mandrelinterface 267 can define an aperture that can receive a pipette mandrel.A skirt 264 may be coupled to the piercing element 266. The pipettemandrel interface, piercing element 266, and the skirt 264 may be asingle unitary piece. In some embodiments, the piercer 262 may comprisean injection molded plastic material or the like. The skirt 264 of thefilm piercer 262 can also act as a contamination cover for the reactionwell.

The film piercer 262 can include a pyramidal blade, with sharp edgesthat slice through the film as it is moved vertically. Other possibleconfigurations, such as a square cross-section or overall conical shapewith a sharp tip are possible. Suitable materials for the film piercer262 may be similar to those noted above for the pipette tips, and caninclude conductive polymers that permit detection by a liquid sensingcircuit. It can also include a handling feature, which is configured tointerface with a pipetting device that is normally used with themillitip.

FIG. 4( f) shows a film piercer 262 as it is used with an assaycartridge 200. As shown therein, the film piercer 262 can pierce abarrier film, and the piercing element can be sized to fit within areagent well. The skirt 262 may have bottom lateral dimensions that arelarger than the area defining the top of the reaction well. As shown inFIG. 4( f), the skirt 262 may allow the piercer to sit on top of thereaction well. In some embodiments, the film piercer 262 can havefeatures that retain it in the assay cartridge 200 during handling,including mechanical features that provide an interference fit, snapfit, or friction fit. The film piercer 262 may also be retained in theassay cartridge 200 using an adhesive.

In use, the film piercer 262 can be manipulated using a pipette mandrel,which is inserted into the pipette mandrel interface 267. In a preferredembodiment, the sample millitip pipettor 704 is used to manipulate thefilm piercer 262. Following acquisition by the pipette mandrel, the filmpiercer is directed downwards at a controlled rate to bring the piercingelement 266 into contact with the barrier film 205 that overlies atleast one of the reagent wells of the assay cartridge 200. In oneembodiment, the barrier film 205 overlaying each reagent well is piercedin one series of operations. In alternative embodiments, the barrierfilm 205 over a portion of the reagent wells may be pierced in oneseries operations and the assay cartridge 200 returned followingintervening steps for piercing of additional portions of the barrierfilm 205.

The film piercer 262 may be disposed of by ejecting it in a mannersimilar to a pipette tip, as described above. In one embodiment, thefilm piercer 262 is ejected into a position on the assay cartridge 200,and is eventually disposed of on disposal of the spent assay cartridge200 following sample processing. In another embodiment, the film piercer262 is disposed of by moving the pipettor carrying the film piercer 262to a designated waste disposal chute that leads to a solid wastecontainer 92. Such a waste chute may be located within the path of thesample pipettor 700. The film piercer 262 may be ejected into this wastedisposal chute by moving a pipette mandrel carrying it through a passivestripping device oriented to direct the film piercer 262 to a wastedisposal chute. This advantageously permits slow and gradual removal ofthe film piercer 262, minimizing the chance of accidental uncontrolledrelease of this sharp device.

FIG. 4( g) shows a portion of an assay cartridge 200 with a vessel base246 and a vessel plug 246 disposed within reaction vessel componentholders 219. FIG. 4( h) shows a top plan view of a cartridge cover 229that is on the assay cartridge 200. FIG. 4( i) is a bottom perspectiveview of the cartridge cover 229. In this embodiment, a cartridge cover229 is present and may be configured to fit on top of the portion of theassay cartridge 200. The cartridge cover 229 may comprise a cover mainportion 229 which may be substantially planar. It may also comprise acover protrusion 229(a) that fits within the vessel base 246 when it ison the assay cartridge 200. As shown, the cartridge cover 229 may extendto the first transverse wall 213 of the assay cartridge 200 to an end ofthe assay cartridge 200, while being laterally coextensive with thelongitudinal walls of the assay cartridge 200. In some embodiments, asimilar cover may be used to protect the reaction well 202 withoutincorporating the piercing function of the film piercer 262.

Referring to FIG. 4( h), the cover protrusion 229(b) can define a hollowrecess 229(b)-1 at the top of the cover 229. The hollow recess 229(b)-1may serve as a handling feature, which can allow a device, such as apipettor or other device to manipulate the cover 229.

Referring to FIGS. 4( g) and 4(i), four corner fitting elements 229(c)can be positioned around the cover protrusion 229(b). This cornerfitting elements 229(c) can be used to position the cover in a vesselcomponent region 231, which may merge into the reaction vessel componentholders 219.

The cartridge cover 229 may be made of any suitable material and mayhave any suitable configuration. For example, it may include anysuitable molded plastic material. It may also include any suitablenumber of protrusions (e.g., two or more), and may have any suitablelateral and longitudinal dimensions.

The cartridge cover 229 may be advantageously used to cover the vesselbase 246 and the vessel plug 246 during processing, so that they areprotected from potential sources of contamination.

Embodiments of the invention may also comprise a pre-cut plasticretention film that surrounds the edges of the compartments holding thereaction vessel base and reaction vessel plug, which provides enoughfriction to hold these items in place during handling while permittingthem to be removed easily using a pipettor mandrel.

E. Reaction Vessel

FIG. 5 shows one embodiment of the invention, which can be directed to areaction vessel 221 for real time PCR. In some embodiments, the reactionvessel 221 can be an amplification vessel, a PCR reaction vessel, or aPCR vessel. The reaction vessel 221 may be sealed or unsealed.Specifically, FIG. 5( a) shows a top perspective view of a reactionvessel 221 according to an embodiment of the invention. FIG. 5( b) showsan exploded view of a reaction vessel according to an embodiment of theinvention. FIG. 5( c) shows a perspective, cross-sectional view of areaction vessel according to an embodiment of the invention.

As shown in FIG. 5( a), the reaction vessel 221 can be a two-partcontainer used to contain the amplification mixture during nucleic acidamplification and detection. Each part resides in a separate compartmentwithin the assay cartridge (see FIG. 4( a)-1). Alternatively, reactionvessel bases 246 and plugs 222 may be provided in racks similar to themicrotip racks 550 described below. The reaction vessel 221 includes avessel base 246 and a vessel plug 222. The system loads the vessel base246 with amplification mixture and then seats the vessel plug 222 ontothe vessel base 246. The amplification mixture may comprise a mixture ofprocessed sample and enzymes, primers, probes, and other materialsneeded for nucleic acid amplification. Once the vessel plug is seated,the vessel plug 222 locks to the vessel base 246 and seals theamplification mixture within the assembled reaction vessel 221. Thereaction vessel 221 can remain sealed and locked through completion ofthe assay to reduce the risk of contamination. In an alternativeembodiment, the vessel base 246 and the vessel plug 222 may be providedas a single unit, with the two portions joined by a flexible tether.

Referring to FIGS. 5( a), 5(b), and 5(c), the reaction vessel 221 caninclude a radially symmetrical reaction base 246, and a vessel plug 222.The reaction base 246 can comprise an upper vessel base portion 246(a)that receives the vessel plug 222 and a lower vessel base portion246(b), which can be a lower portion of the vessel base. The lowervessel base portion 246(b) opens into the upper (cylindrical) vesselbase portion 246(a) and comprises a frustum of a conical shape. Theterms “lower” and “upper” can refer to the relative positions of theportions of the vessel base, when the vessel base is used in the system.The vessel plug 222 may also include a handling feature 222(f). Thehandling feature 222(f) can comprise a cylindrical enclosure configuredto receive a pipette mandrel (not shown).

The symmetrical nature of the base 246 can allow the system to place thereaction vessel in an arbitrary orientation about the axis of thereservoir region. That is, when a radially symmetric vessel is placedinto a complementarily shaped cavity, unlike a vessel with a rectangularcross-section, it does not matter how the vessel is oriented, as long asthe primary axes of the reaction vessel and the cavity are aligned.

The reaction vessel 221 may include any suitable number or types ofdistinct features or materials. For example, the material forming thebase 246 and/or plug 222 may include a material that has the followingcharacteristics: a thermal conductivity greater than about 0.1 W/m·K; aYoung's modulus of about 1.5 GPa to about 2 GPa; and a frictionalcoefficient of less than about 0.25. The material may comprise a polymersuch as polypropylene and it may have elastomeric properties with ahardness ranging from 20 to 50 durometer (Shore) A, and it may also beconductive. In one embodiment, the polymer has a hardness of about 30durometer (Shore) A. Suitable materials for the vessel base can betransparent as well as translucent. Other suitable alternative materialsfor the vessel base may include polyethylene, polystyrene, polyacrylate,polycarbonate, silicone, and copolymers and blends thereof.

The base 246 may also include any suitable geometry or features. Forexample, in some embodiments, the lower vessel base portion 246(b) ofthe vessel base 246 has a geometry where the walls (in a cross-sectionalview as shown in FIG. 5( c)) form an angle between about 4 degrees andabout 8 degrees, or about 6 degrees. Further, the lower vessel baseportion 246(b) may include a volume of about 10 μL to about 70 μL, and aterminus of the lower portion 246(b) of the reaction vessel base 246 canhave an optical window. A wall thickness of the lower portion 246(b) maybe about 0.0005 inches to about 0.02 inches.

In some cases, the upper vessel base portion 246(a) comprises a latchingfeature 246(a)′ that engages the plug 222 on insertion, so that thelatching feature 246(a)′ irreversibly secures the plug 222. In thisexample, the latching feature 246(a)′ may be the latching portion. Theplug 222 can form a seal that is resistant to a pressure of at leastabout 50 psi when the plug 222 is engaged in the upper vessel baseportion 246(a) of the reaction vessel base 246. The latching feature246(a)′ of the upper cylindrical portion can comprise one or a pluralityof flexible locking tabs, which may be in the form of ridges, where theflexible locking tabs project downwards and centrally, displace outwardson initial insertion of the plug 222, move centrally on seating of theplug 222 in the reaction vessel base 246, and engage the plug 222 onmoving centrally.

The latching feature 246(a)′ of the upper cylindrical portion 246 cancomprise a circumferential ridge, where it projects centrally. It mayalso expand radially on initial insertion of the plug 222. It may alsocontract radially on seating of the plug 222 in the reaction vessel base246. The circumferential ridge engages the plug on radial contraction ofthe upper cylindrical portion of the reaction vessel base 246. Thelatching feature 246(a)′ of the upper vessel base portion 246(a) canalso comprise a plurality of arcuate ridges, wherein the arcuate ridgesproject centrally. The upper vessel base portion 246(a) of the reactionvessel base 246 can expand radially on initial insertion of the plug222, can contract radially on seating of the plug 222(f) in the reactionvessel base 246, and can engage the plug 222 on radial contraction ofthe upper vessel base portion 246(a) of the reaction vessel 221.

In some embodiments, plug 222 comprises a block of elastomer with adiameter greater than that of the opening of the lower portion of thereaction base. It may also include a handling feature 222(f), which maycomprise an inner surface, an outer surface, and a longitudinal groove222(e). A cylindrical enclosure of the handling feature 222(f) can havean internal diameter of about 0.125 to about 0.4 inches in someembodiments, and the inner surface of the cylindrical enclosure cancomprise a plurality of projections 222(d) (such as protrusions), whichcan be hemispherical.

The vessel base 246 can be further characterized to include a reservoirregion and a locking region. The locking region may correspond to theupper vessel base portion 246(a), while the reservoir region maycorrespond to the lower vessel base portion 246(b). The reservoir regionholds the amplification mixture, and the locking region cooperates tolock and retain the vessel plug 222 once seated.

The vessel base 246 may be made of any suitable material. A suitablematerial for the vessel base 246 is a translucent polymer capable ofwithstanding the elevated temperatures and pressures of theamplification process and compatible with its chemical conditions.Suitable materials include PD702 polypropylene homopolymer manufacturedby LyondellBasell Industries of Rotterdam, The Netherlands.

The reservoir region corresponding to the lower vessel base portion246(b) may include a thin-walled, truncated cone that holds up to about50 microliters of amplification mixture. In some embodiments, thereservoir region is a frustum of a cone, a shape that serves to improvethermal contact between the reservoir region and a thermal cycler heatblock. The conical shape improves thermal contact with a complementarilyformed region of the heat block both at the macroscopic scale and at themicroscopic scale. At the macroscopic scale, the conical shape reducestolerance requirements by using a single extended surface for alignment.At the microscopic scale, the conical shape permits simple downwardpressure to increase asperity contact over the full surface. Improvedthermal contact decreases the response time to temperature changes andhence decreases the length of each thermal cycle. Shorter thermal cyclelength may have a beneficial effect on the total time to produceresults, as the thermal cycle may repeat many times during each assay.

The reservoir region's conical shape may have a small opening angle.That is, the sides are close to parallel with the axis of the reservoirregion. A small opening angle provides a conical volume where eachelement of volume along the axis is relatively equidistant from thenearest wall. Since the elements of volume along the axis are the mostdistant from the walls, and since thermal transfer decreases withdistance, these elements are the last to reach target temperature. Asmall opening angle improves temperature uniformities along the axis byassuring that each axial fluid element has about the same thermaldistance from the wall as each other axial fluid element. Improvedtemperature uniformity may directly contribute to assay precision byreducing variations between regions in the amplification mixture. Insome embodiments, the opening angle is less than about 15 degrees and insome cases is about 6 degrees.

In some embodiments, a substantially flat bottom surface truncates theconical portion of the reservoir region. The flat bottom portion of thereservoir region can be an optical window that can be used formonitoring or characterizing vessel contents. For example, the flatbottom may be an optical window for excited or emitted light to enterthe reaction vessel or for emitted light to leave the reaction vessel221. In some embodiments, the circumferential edge of the bottom of thereservoir region extends slightly beyond the exterior surface of theflat bottom to recess the bottom surface. The recessed surface mayreduce the likelihood of damage to the optical window in handling.Alternatively, the bottom surface may curve to act as the boundingsurface of a lens. Such a lens may focus light in a desired patternwithin the reaction vessel 221 or may enhance collection of light fromwithin the reaction vessel in embodiments where the optical windowcollects emitted light from the reaction vessel 221.

The reservoir region can be thin-walled to support intimate thermalcontact between the reservoir region contents and external heaters. Likethe bottom portion of the reservoir region, the side walls of thereservoir region can also be an optical window that can be used formonitoring or characterizing vessel contents. In some embodiments, thereservoir region wall thickness is as thin as practical based onstrength of the materials used, on production process considerations,and on uniform clarity. The wall material can be strong enough towithstand elevated pressures and temperatures during amplification. Thewall may soften and deform during amplification, possibly causing it toconform to and adhere to the thermal cycler heat block. The wallmaterial can have sufficient strength such that, once so deformed, thesystem may detach the reaction vessel from the heat block withoutrupturing the reaction vessel 221.

As noted above, since the system samples emitted light through the wallof the reservoir region, any portion of the wall in a band at the lightsampling height may act as an optical window. The production processcontrols mold filling to maintain optical uniformity throughout thisband. Using injection molded polypropylene, the reservoir region wallthickness can be less than about 0.50 mm and in some cases about 0.10mm, or less.

In some embodiments, amplification monitoring involves providingexcitation light illuminating the amplification mixture and detectingemitted light that the amplification mixture produces in response to theexcitation light. At least the vessel base 246 is at least partiallytransparent or translucent to both excitation light and emitted light inorder to allow monitoring of amplification progress. Both excitation andemitted light need to traverse the vessel base wall; the translucentnature of the vessel base 246 wall makes this possible. Any othersuitable part of the reaction vessel may also be transparent ortranslucent.

Additional considerations relevant to wall material selection includechemical compatibility, cleanliness, compliance, and cost. Wallmaterials can be chemically compatible with reaction conditions. In someembodiments, wall materials have at least some compliance to improvethermal contact when pressed into the thermal cycler. Such compliancemay also help in locking the vessel base to the vessel plug. A varietyof polymers, including polyolefins, polystyrene, PEEK, fluorocarbonpolymers, and other polymers may be suitable. In a preferred embodiment,the wall material is polypropylene. Reaction vessel materials can befree of contaminants that might interfere with amplification ordetection reactions. This may be accomplished by using only virginmaterials in the manufacture of the reaction vessel, by eliminatingunprotected handling of either reaction vessel components or ofequipment used in their production, and by treatment of equipment withmaterials that destroy potential contaminants. In some embodiments, thevessel may comprise a polymer such as PD702, which is a high flow,controlled rheology polypropylene homopolymer resin.

Other embodiments of the invention are directed to the process of makingthe reaction vessel. In some embodiments, the reaction vessel base ismade by injection molding. This is usually performed by injecting theplastic at a position where the thickness of the molded part is greatestand allowing it to flow to where the thickness is least, however, thethin sections may produce high resistance to molten polymer flow inplastics injection molding. Such high flow resistance may contribute toincomplete filling, particularly when parts mix thick and thin sections.The reaction vessel can be formed by injecting the fluid plastic througha gate corresponding to the lower terminus of the vessel, where thewalls are thinnest. This avoids problems often seen in conventionalinjection molding methods, where thin sheets of rapidly cooling plasticfail to blend completely and form partially opaque or mechanically weakareas.

The locking region of the vessel base 246 connects to the reservoirregion and is annularly disposed upward and outward of the reservoiropening. In some embodiments, the locking region and the reservoirregion form a single integrated part made of a single material in asingle forming process. The locking region may include a plug receivingportion 251, a sealing portion 252, and a latching portion 250.

The sealing portion 252 extends upward and outward from the reservoirregion, connecting the reservoir region to the plug receiving portion251 of the locking region. The sealing portion 252 acts as a transitionto the larger diameter plug receiving portion 251 and provides a sealingsurface for the vessel plug 222 to seal against. The sealing portion 252may form a conical annulus flaring out from the reservoir region andcontinuing as the walls of the plug receiving portion. The internalangle of the conical annulus is greater than 90 degrees and in somecases about 120 degrees. In some embodiments, the sealing portion 252has thicker walls than the reservoir region to resist deformation whilesealed. In some cases, the sealing portion walls are about twice thethickness of the reservoir region walls. The sealing portion 252 mergesinto the reservoir portion in a smooth transition, including a slightoverhang so that the diameter of the opening in the sealing portionannulus is smaller than the diameter of the upper portion of thereservoir portion. The overhang can be sufficiently small such that theproduction process may “bump” the parts from the mold. In someembodiments, the overhang is less than about 0.1 mm and in some casesabout 0.06 mm. This overhang advantageously deforms the elastomeric sealof the vessel plug 222 to more tightly seal the reaction vessel.

The seal made by the plug 222 when it is inserted into the vessel 221can be characterized as a hybrid seal, having characteristics of both aradial seal (such as an O-ring) and a face-to-face seal (where a seal issimply pressed against a surface).

The plug receiving portion 251 of the locking region may extend upwardsfrom the sealing portion 252 to form a roughly cylindrical segmentcoaxial with the reservoir region and with the sealing portion 252. Thesegment may taper outward towards the top for easy mold release. Thepurpose of the plug receiving portion 251 is to connect the sealingportion 252 to the latching features and to retain the plug body portionof the vessel plug 222. The distance between the portion of the vesselplug 222 that engages the latching portion 250 and the vessel plug'selastomeric seal determines the length of the plug receiving portion251; the plug receiving portion 251 can be long enough to allow thevessel plug to engage into the locked position with sufficientcompression of the elastomeric seal to adequately seal the reactionvessel 221. The plug receiving portion 221 couples at its top to thelatching portion.

The latching portion 250 cooperates with engagement features on thevessel plug 222 to lock and retain the vessel plug 222 to the vesselbase 246. The latching portion 250 may extend outward and upward fromnear the top of the plug receiving portion 251 as a base flangeconnecting to a substantially cylindrical side wall. The side wall mayextend slightly below the base flange. Vertical cuts may divide thecylindrical side wall into two or more sections to increase radialflexibility. In some embodiments, three vertical cuts divide thecylindrical side wall into three symmetrical sections. Each section mayinclude a circumferentially disposed medial portion flanked bysymmetrical lateral portions. Each medial portion may include a latchingfeature 246(a)′ projecting inward from the cylindrical side wall. Theupper surface of the latching feature 246(a)′ can slope downward towardsthe part center to allow the vessel plug 222, as it enters, to deflectthe cylindrical side wall outward. The lower surface of the latchingfeature 246(a)′ is substantially perpendicular to the axis of the vesselbase 246. Once the engagement feature (corresponding to the vessel thirdplug portion 222(c)) of the vessel plug 222 descends below the lowersurface of the latching feature 246(a), the cylindrical side wallrecovers by snapping back towards the centerline. This snap back actiontraps the engagement feature of the vessel plug 222 beneath the lowersurface of each latching feature 246(a)′. In an alternative embodiment,the cylindrical side wall of the latching portion 250 is not divided byvertical cuts, and a circumferential ridge extends medially to form anannular latching feature. In a second alternative embodiment, thecylindrical side wall of the latching portion 250 is divided into aplurality of symmetrical sections, each section having a latchingfeature 246(a)′ that is continuous with the upper rim of the cylindricalside wall and that extends both medially and towards the vessel base246. These latching features 246(a)′ are deflected outwards as thevessel plug descends through the latching portion, and recover bysnapping back towards the center line as the vessel plug descends belowthe lower surface of the latching feature. This snap back action trapsthe engagement feature of the vessel plug 222 beneath the lower surfacesof the latching features 246(a)′.

Relief openings can pierce the flange connecting the side wall to theplug receiving portion 261. The relief openings underlie each latchingfeature 246(a)′ to prevent undercuts in the vessel base and avoid morecomplex mold operations. The remainder of the flange connects to theside wall and may continue as the lateral portions of the side wallsections. These lateral portions provide stiffness to produce the snapback action that engages the vessel base 246 to the vessel plug 222.

In some embodiments, the upper opening of the latching portion 250includes an inward and downward facing chamfer contiguous with the uppersurface of the latching feature. This chamfer helps to center the vesselplug 222.

The vessel plug 222 closes and seals to the vessel base to retainreaction vessel contents. This seal may be resistant to pressures of upto 50 pounds per square inch. Retention is desirable both to preventevaporative loss that might alter concentrations during amplificationand to prevent amplified nucleic acid from contaminating other assays.In an embodiment, the vessel plug 222 includes an elastomeric seal and aplug body that supports the elastomeric seal. In an alternativeembodiment, the sealing surface of the reaction vessel base 246incorporates an elastomeric O-ring and the seal is formed by closure ofthe vessel plug 222 against this O-ring. In another embodiment, thesealing surface of the reaction vessel base 246 and the vessel plug 222have a friction fit on insertion of the vessel plug 222, the frictionfit forming a seal. In another embodiment, the reaction vessel base 246and the vessel plug 222 incorporate collapsible seal regions that form aseal on insertion of the vessel plug 222 into the reaction vessel base246. Vessel plugs 222 may be at least partly opaque to excludeinterfering light during processing.

In some embodiments, the plug body is electrically conductive. This hasthe benefit of supporting measurements by a sensing circuit, such as aliquid sensor. Such a sensing circuit may be associated with a pipettorthat is used to transport a vessel plug 222, advantageously providing ameans to verify acquisition of the vessel plug 222 or signal loss duringtransport. A preferred method of producing electrical conductivity inthe plug body is admixture of a base polymer with a conductive materialsuch as carbon or metallic particles.

For some embodiments, the elastomeric seal can be a thermoplasticelastomer with hardness of 30-40 durometer (Shore) A. In otherembodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A.Elastomers deform sufficiently to form a tight seal with the vesselbase. Thermoplastic elastomers are advantageous because of theircompatibility with plastics injection molding processes.

The vessel plugs 222 can be formed in any suitable manner. In someembodiments, the forming process for vessel plugs is two-part plasticsinjection molding. The process overmolds the elastomeric seal about thepre-formed plug body. The polymer for the elastomeric seal can beinjected into the same mold while the polymer for the plug body is stillin place and warm, allowing the two polymers to flow into each other andfor chemical bonds that hold the elastomeric seal firmly in placewithout adhesive. This has the advantage of producing high quality partsat relatively low expense. In some embodiments, the molding processforms the plug body of a carbon-loaded polypropylene such as RTP 199 X106053A produced by RTP Company of Winona, Minn. The preferred materialfor the elastomeric seal is Dynaflex™ G7930-1001 produced by PolyOneCorporation of McHenry, Ill.

In some embodiments, the elastomeric seal may be substantiallycylindrical with a chamfered lower end. The upper end may extend into aretaining aperture at the bottom of the plug body, such that the upperend infiltrates into the retaining aperture, capturing the elastomericseal to the plug body. In some embodiments, the retaining aperture iscounterbored with the larger diameter distal from the bulk of theelastomeric seal so that the elastomer may expand into the counterborefor better retention. Alternatively, the manufacturing process may formthe elastomeric seal as a separate piece from the plug body and may bondthe elastomeric seal to the plug body through another method such as afriction fit or an adhesive.

The elastomeric seal can be large enough to provide adequate compressionwithout bottoming on the sealing portion of vessel base 246. Thehardness and dimensions can cooperate to allow the elastomeric seal tothe sealing portion with reasonable sealing force. In some embodiments,the elastomeric seal diameter is small enough so that, when compressedby engagement of the vessel plug to the vessel base, it conforms to thesealing portion without contacting the internal wall of the plugreceiving portion. This advantageously concentrates sealing force to thesealing portion of vessel base and distributes sealing force evenly toprevent leaks. In some embodiments, the sealing force is about 44newtons (about 9.9 lbs) and produces a pressure on the sealing surfaceof about 300 (about 43.5 pounds per square inch) to about 1000 kPa(145.0 pounds per square inch).

The vessel plug 222 has functions of supporting the elastomeric seal,engaging with the latching features 246(a)′ of the vessel base 246,transmitting the engagement forces from the latching features 246(a)′ tothe elastomeric seal, mating to a pipettor mandrel for handling, andindicating successful mating. The vessel plug 222 may comprise asubstantially cylindrical tube supporting the elastomeric seal at thetube's bottom. A portion of the vessel plug 222 may extend above the topof the reaction vessel 221 when it is engaged, in order to ensure thatseating forces applied by the compression head 1342 of the slidable lid1315 (see FIG. 16( i)) are transmitted through the vessel plug andfurther securing the elastomeric seal during thermal cycling. In someembodiments, two or more vertical plug slots split the plug body for afraction of the plug body length to provide radial flexibility. The tubeterminates at the top end in one or more engagement features. Theengagement features may correspond to parts of a vessel plug thirdportion 222(c). As shown in FIG. 5( c), the vessel plug third portion222(c) may be coupled to a vessel plug first portion 222(a), and avessel plug second portion 222(b). The first, second, and third vesselplug portions 222(a), 222(b), 222(c) may all be integrally formed withrespect to each other.

The engagement features engage the latching features 246(a)′ on thevessel base 246 to seat the vessel plug 222 to the vessel base 246. Onceseated, in some embodiments, the system does not remove the vessel plug222, nor is the plug 222 designed to be removable by a user. The intentis to join the parts together and seal the reservoir region for at leastas long as the parts remain in the system and as long as the partsremain anywhere within the laboratory. This advantageously preventsescape of any amplified nucleic acids that might otherwise contaminateassays or samples. In some embodiments, the engagement features aresegments of a locking flange extending outward from the top end of theplug body. The height of the locking flange is slightly less than thegap distance of the vessel base 246 between the upper surface of thebase flange and the lower surface of the latching feature. The lockingflange thus fits within the gap distance. Compliance of the parts,particularly the compliance of the elastomeric seal, may take upvariations within the component production tolerances.

The plug body may also include a circular locating wall 222(g) extendingupward from the upper surface of the locking flange with outer diametercomplementary to the inner diameter of the latching features 246(a)′ ofthe vessel base 246. The locating wall advantageously limits relativemotion of the vessel plug and the vessel base to maintain the seal. Twoor more vertical flange slots may segment the locking flange and thelocating wall. These flange slots continue as the plug slots thatsegment a portion of the plug body and provide flexibility to the part.The vessel plug 222 may also include a counterbored aperture asdescribed above.

The engagement features lock to the complementary latching features246(a)′ on the vessel base 246 and dispose the elastomeric seal insealing contact with the vessel base sealing surface. As noted above,the engagement features may correspond to parts of a vessel plug thirdportion 222(c). Compliance of the elastomeric seal pushes the vesselplug upwards so that the upper surface of the locking flange on thevessel plug 222 contacts the lower surface of the latching feature onthe vessel base 246. The efficacy of the sealing contact depends oncooperation of several dimensions and material properties, but a widevariety of dimensions may still achieve acceptable sealing contact. Somedimensions may change together without affecting the seal. For example,a longer plug body matched with a longer plug receiving portion wouldhave only minor effect on the seal efficacy. Similarly, a softerelastomeric seal might compensate for a longer plug body or a stifferbody plug material might work with a shorter plug body. In someembodiments, interaction with other parts at least partly determines thelength of the plug body, and this length in turn may determine the sizeof the plug receiving portion. Commercial availability of usefulthermoplastic elastomers at least partly determines the elastomeric sealhardness. The primary determinant of the dimensions and materialproperty combination is that the combination provides the requiredsealing efficacy.

The plug body interior can accept and grip a pipettor mandrel to allowthe system to move the vessel plug or the closed reaction vessel. Theplug body inside diameter may be slightly smaller than the mandreloutside diameter, but the plug slots permit the plug body to flex andexpand radially as the mandrel enters. The plug body length, materialstiffness, and plug slot length cooperate to open the plug body withreasonable downward force of the mandrel and to provide adequategripping strength. The restoring force of the flexed plug body serves togrip the mandrel. Additional geometry within the plug body may serve toenhance gripping. In some embodiments, four hemispherical protrusions ofthe tube wall material into the lumen of the plug body help to grip thepipettor mandrel. These protrusions advantageously concentrate therestoring force to produce a high pressure contact with the mandrel.High pressure contact increases the friction between mandrel and plugbody to better retain the plug body on the mandrel. The plug 222 may bemade of conductive plastic, allowing the plug to be detected by thepipettor with a suitable sensing circuit, such as a liquid sensor.Alternatively a pipettor may detect the presence of a vessel plug 222using a pressure sensor that measures pressure within the pipettor,generating a pressure profile that is characteristic of the presence ofa vessel plug 222 on the pipette mandrel. In some embodiments both aliquid sensor and a pressure sensor are used to detect the presence of avessel plug 222 on a pipettor.

FIG. 5( d) shows a perspective view of a reaction vessel 221 accordingto another embodiment of the invention. The configuration of thereaction vessel 221 shown in FIG. 5( d) is generally similar to theconfiguration of the reaction vessel shown in FIGS. 5( a)-5(c). In theembodiment shown in FIG. 5( d), the plug 222 now has a rim that extendsup over the edge of the vessel base 246 when inserted. This ensures thatwhen the slidable lid of a thermal cycler module is closed, it pressesdown on the plug 222, securing it tightly. The upper surface of the plug222 may extend any suitable distance (e.g., at least about 1 mm) abovethe top surface of the vessel base 245 when the plug 222 is insertedinto the vessel base 245.

Alternative reaction vessel embodiments include a vessel that is made ofa flexible material that conforms to the shape of the heating block,rather than a pliant material that only bends slightly. In yet otherembodiments, the vessel can have a non-circular cross-section, havingconfigurations that are wedge shaped, rectangular, or polygonal.

The above-described reaction vessel can be used in a process fordetermining a nucleic acid in a sample using a system including apreparation location and a thermal cycler module. The process caninclude providing in the preparation location a vessel plug with ahandling feature and a vessel base configured to lockably engage withthe vessel plug; pipetting an amplification reagent to the vessel basewith a pipette tip held on a mandrel; pipetting the nucleic acid to thevessel base; lifting the vessel plug using the mandrel to grip thehandling feature; engaging the vessel plug to the vessel base; andmoving the engaged vessel plug and vessel base to the thermal cyclermodule. Each of the features of this process is described in furtherdetail above and below. This and other processes described herein canprovide for efficient processing of nucleic acids, since a pipettemandrel can be used to perform multiple functions.

F. Millitip

Embodiments of the invention can also include the use of millitips.

FIG. 6( a) shows a millitip according to an embodiment of the invention.FIG. 6( b) shows a mounting aperture of a millitip. FIG. 6( c) shows amillitip on a mandrel.

In embodiments of the invention, a millitip 220 can be a relativelylarge capacity pipette tip carried within each assay cartridge and usedduring the isolation phase. Multiple processes within the system may usethe millitip, but the system can use each millitip for transfersinvolving a single assay cartridge. In some cases, each millitip is onlyused for transfers involving a single assay cartridge. This reduces thepossibility of inter-sample contamination. In some embodiments, themillitip has a capacity of at least one milliliter and tapers to apipetting orifice. Millitips may couple to pipettors through a compliantcoupling taper that supports repeated remove and replace operations.Length of the millitip may be sufficient to reach the depth of a 100 mmtube or other sample containers used on the system when mounted on asuitable pipette mandrel. Millitips may incorporate barrier and ventingfeatures. Preferred materials are electrically conductive non-reactivepolymers.

As shown in FIGS. 6( a)-6(c), the millitip 220 can be a generallyconical hollow body open at both ends with axial symmetry. The centrallumen opens into a pipetting orifice 220(b) at the millitip apex andinto a mounting aperture 220(f) at the millitip base. The mountingaperture 220(f) couples to a pipettor mandrel during use; pipettedfluids enter and leave through the pipetting orifice 220(b).

In some embodiments, the walls forming the millitip 220 are thin andtapered; the walls may be about 0.8 mm thick near the base and about 0.5mm thick at the apex. The wall thickness can be sufficient to give themillitip 220 mechanical strength sufficient for penetrating barrierfilms on containers, or to open valves (e.g., a “duckbill” valve of acovered tube). The conical body may taper in several segments. Segmentedtapering advantageously permits a narrow pipetting orifice to couple toa large capacity pipette tip. The large capacity pipette tip supportssingle step transfer of reagents, saving time and improving transferprecision. The narrow pipetting orifice supports good transferprecision, which directly improves assay precision. The intermediatetapers permit both high capacity and good precision in a pipette tip ofpractical length.

In some embodiments, a coupling taper 220(a) extends from the mountingaperture 220(f) to a lower diametral step forming a seating surface220(a)-2. In other embodiments, the seating surface can be ribs or otherprotrusions that extend slightly from the end of the tip. The millitip220 continues below the seating surface as an upper taper that extendsfor the majority of the part length. A lower taper 220(c) forms theapical end of part that in some embodiments terminates in a 1.3 mmdiameter flat annulus surrounding a 0.8 mm pipetting orifice. Theannulus is disposed perpendicular to the long axis of the millitip 220.A middle taper 220(d) connects the lower taper 220(c) and upper taper220(e). The millitip walls can be of constant thickness (about 0.8 mm)through the entire part, except for the lower taper 220(c) and themounting aperture 220(f). Walls of the lower taper 220(c) may thin outtowards the apex. The interior taper angles defining the lumen mayincrease in steps towards the apex. The angles can be about 0.8 degreesin the coupling taper, about 2.8 degrees in the upper taper 220(e),about 3.2 degrees in the middle taper, and about 6.0 degrees in thelower taper 220(c) (all measured with respect to the millitip axis).

The coupling taper 220(a) may be a compliant taper with a smoothinterior surface and without supporting ribs. The absence of ribsincreases compliance and contributes to a smooth inner surface inplastic injection molded parts by eliminating sink marks associated withvariable thickness sections. Thinner walls (about 0.45 mm) contribute toincreased compliancy in the coupling taper. Compliancy in the couplingtaper 220(a) has the benefit of allowing a millitip to elasticallydeform with minimal resistance when coupling to a pipetting mandrel.Elastic deformation advantageously permits recovery to near the originalshape, permitting the system to load and unload the millitip fromseveral different pipettor mandrels while preserving a fluid tight sealon each use.

In some embodiments, the coupling taper 220(a) may abruptly changediameter at the top of the upper taper forming a seating surfaceperpendicular to the axis of the millitip 220. This seating surface mayrest on a complementary surface in the assay cartridge and support themillitip 220 at a controlled height and within a controlled locus. Insome embodiments, interaction of the millitip 220 at the height of theseating surface and the assay cartridge control the locationsufficiently to permit lead in features on the descending pipettormandrel to align the millitip 220 with the mandrel during millitippickup. In some embodiments, the seating surface forms a flat annulusabout 0.7 mm wide surrounding a 7 mm core.

The open end of the coupling taper 220(a) forming the mounting apertureends in a stopping annulus 220(a)-1 disposed perpendicularly to the axisof the millitip 220. The stopping annulus may interact with features ona pipettor mandrel to provide a fixed relationship between the height ofthe mandrel and the height of the millitip 220. This advantageouslylocates the pipetting orifice with respect to the controlled height ofthe pipettor to more precisely aspirate, dispense, and mix liquids.

Millitips may incorporate an aerosol barrier 220(h). In someembodiments, the upper taper 220(e) section of the millitip 220 includesan abrupt internal diametral decrease slightly below the seating surface(with the millitip oriented in the normal operating position with thepipetting orifice at the bottom). This diametral decrease forms a stepthat may retain a self-supporting porous substrate as an aerosolbarrier. The aerosol barrier reduces the likelihood of contaminationduring pipetting by preventing any aerosols or splashes escaping the topof the millitip 220.

Millitips can incorporate venting features. Venting features may serveto equalize pressure in a reagent well as a millitip aspirates ordispense the contents of a reagent well through a compliant barrierseal. Since such a barrier film may effectively seal around a millitip,the pipetting operation may change the pressure in the reagent well. Achange in reagent well pressure may affect pipetting precision orgenerate aerosols that are a potential source of contamination. Ventingfeatures advantageously maintain a patent air flow path through thebarrier film while the millitip is in the well. This patent air flowpath allows more rapid pressure equalization across the barrier film,which reduces the effect of barrier film interference with pipettingprecision. Improved pipetting precision may directly improve assayprecision. In some embodiments, venting features include abruptdeviations from the millitip's otherwise smooth conical outside wall.Such deviations can extend in the vertical direction so as to at leastoverlap the location of the barrier film during pipetting. Ventingfeatures may include sharp corners on the outside diameter, protrudingribs, incised channels, or similar features. In addition, the exteriorof the millitip pipette orifice may be an annulus, the plane of which isat right angles to the central axis of the millitip. Such aconfiguration prevents a tight seal from forming when the lower terminusof the millitip is in contact with the bottom of an angled well, such asthe reaction well of the assay cartridge, thus improving pipettingaccuracy.

In some embodiments, millitips are electrically conductive. This has thebenefits of dispelling the effects of static electricity and supportingmeasurements by a sensing circuit, such as a liquid sensor, as describedin more detail below. Static electricity may cause lightweight partswithout a discharge path to accumulate charge causing unfavorableinteractions with other structures. For example, a pipette tip thatacquires a charge (as by sliding engagement with a pipettor mandrel) mayrepel other charged pipette tips to such an extent that charged tips aredisplaced from known locations. The displaced tips may becomeunavailable for use and may interfere with other mechanisms. A preferredmethod of producing electrical conductivity in millitips is admixture ofthe base polymer with a conductive material such as carbon or metallicparticles.

It is also noted that measurements by a sensing circuit associated witha pipette mandrel can be used to indicate successful attachment of theconductive tip to the mandrel, and detachment of the conductive tip fromthe mandrel. It can also permit liquid sensing via the mandrel throughthe conductive pipette tip, and provide an indication of the fill levelof a conductive pipette tip that is carrying liquid. A sensing circuitis described in further detail below.

The preferred forming process for millitips is plastics injectionmolding. This has the advantage of producing high quality parts at lowexpense. In some embodiments, the molding process forms each millitip ofa carbon-loaded polypropylene such as RTP 199 X 106053A produced by RTPCompany of Winona, Minn.

G. Cartridge Loading Unit

FIG. 7( a) shows a top perspective view of an assay cartridge loadingunit.

FIG. 7( b) shows a partial top perspective view of an assay cartridgeloading unit.

FIG. 7( c) shows a perspective view of an assay cartridge presentationlane of an assay cartridge loading unit.

The assay cartridge loading unit 112 serves as an area for loading andtemporary storage of assay cartridges 200 on the system. In operation,the operator may load fresh assay cartridges 200 into the system at thecartridge loading unit 112, also called the CLU 112, withoutinterrupting normal instrument operation. After loading, the CLU 112 mayread identifying indicia, such as a barcode, that are attached to theloaded assay cartridges. The assay cartridge 200 may then be transportedto allow addition of sample from the sample pipettor 700 and processingby an XYZ transport device (described in further detail below). The CLU112 may then transfer the assay cartridge 200 to the transfer shuttle898 (shown in FIG. 14) for further processing.

As shown in FIG. 7( a), the CLU 112 can include two subassemblies: anonload module 119 and a presentation lane 113. In some embodiments, theCLU 112 can have a movable gate (not shown) that can selectively preventcartridges from moving from an onload lane of the onload module 119 tothe presentation lane 113. This gate can be pneumatically actuated. Anaccess door (not shown) can also be provided to provide other access tothe CLU 112. Power to the onload lane motors of the CLU can be cut whenthe access door is opened, as a safety feature.

These two subassemblies may be separate until assembled onto the mainsystem. The onload module 119 may be coupled to and orientedperpendicular to the presentation lane 113. The presentation lane may bean example of a loading lane. Assay cartridges 200 can be loaded intothe onload module 119. In one embodiment, the onload module 119 caninclude a storage location comprising a cavity configured to hold assaycartridge 200. This cavity may be embodied as the interior space of acartridge lane. In some embodiments, the storage location comprises twoonload cartridge lanes (112(b) and 112(c)) that hold one or more assaycartridges each, an interlocked cover 112(a) (which may be a hinged orslidable cover), a touch pad for operator interaction, and a barcodereader (not shown). Although the storage location comprises twocartridge lanes in this embodiment, in other embodiments, the storagelocation may comprise only one, or even three or more cartridge lanes.In embodiments of the invention, the sliding cover of the CLU is lockedunless the onload lanes are idle and the movable gate is closed toprevent the operator from accidentally forcing assay cartridges into thepresentation lane while loading. Embodiments of the invention can alsoprevent jamming.

Each lane can include a CLU baseplate 118 for supporting and aligningcomponents of the cartridge loading unit 112, CLU rails 122 that theassay cartridges rest upon, a loading transport such as a pusher 112(d)mounted to a linear rail, and sensors that detect the presence of theassay cartridges 200. These sensors may be optical, electrical,magnetic, or electromagnetic sensors. Although the loading transport inthis embodiment is a pusher, in other embodiments, the loading transportcould be a device that pulls the assay cartridges towards the cartridgepresentation lane.

The pushers 112(d) can be driven by a stepper motor and belt in a mannersimilar to the previously described pusher plate 617 of the samplepresentation unit 110, and may have a home position within the onloadmodule 119. The stepper motor may have an encoder. The system candetermine the number of assay cartridges loaded by packing thecartridges using the pusher and using the encoder position. Any suitabletype of encoder may be used

In one embodiment, the onload module 119 may be temperature controlled.Temperature control of the onload module 119 may be achieved by theinclusion of thin film heaters, infrared emitters, thermoelectricdevices, a flow of heated or refrigerated air through the unit, or othermeans. Temperature control devices may be incorporated into or affixedto a portion of the CLU baseplate 118 that is adjacent to the onloadlanes (112(b) and 112(c)). Different portions of the CLU may bemaintained at different temperatures. Although two onload lanes 112(b),112(c) are shown, it is understood that embodiments of the invention mayinclude any suitable number of onload lanes.

The onload module 119 can also include a cover sensor and latch. Thelatch locks the cover 112(a) and is unlocked when the pusher 112(d)moves past a designated position. Alternatively, the latch may be movedto the locked and unlocked position using a linear actuator, pneumaticcylinder, or solenoid. In one embodiment, as a safety feature when thecover (112(a)) is opened the cartridge sensors lose power.

In operation, the user may load assay cartridges 200 into the CLU (112)as follows:

(a) The user signals their intention to add assay cartridges 200 to theCLU 112 by pressing a “load” button (physical or virtual).(b) The system waits for the onload lanes to become idle.(c) The movable gate between the onload and presentation lanes closes.(d) The CLU pushers 112(d) move to their home positions.(e) The CLU pusher 112(d) in the front cartridge lane 112(c) moves to adesignated Open Cover position to unlock the cover 112(a).(f) The user opens the cover 112(a), adds assay cartridges 200 to one ormore cartridge lanes (112(b) and 112(c)), and closes the cover 112(a).(g) CLU pusher 112(d) in the front cartridge lane 112(c) returns to thehome position to re-lock the cover 112(a).(h) The pusher (112(d)) moves to the assay cartridges 200 forward untilthey stall against the movable gate.

In some embodiments, each lane (112(b) and 112(c)) of the cartridgeloading unit 112 can hold up to 50 assay cartridges 200. In otherembodiments, more the number of assay cartridges held by each lane canbe more or less than 50. In some embodiments assay cartridges can beloaded into a cartridge lane by using a magazine of cartridges, ratherthan individually.

As shown in FIG. 7( b) each cartridge lane (112(b) and 112(c)) can beconfigured to hold specific types of assay cartridges 200. In oneembodiment, one type of assay cartridge 200 is used for DNA isolationand a second type of assay cartridge 200 is used for RNA isolation. Thisconfiguration can be added or changed by a user. For example, if theoperator generally studies only DNA samples, both cartridge lanes(112(b) and 112(c)) can be configured for DNA assay cartridges 200.Configuration can be performed by attaching an identification bar 112(f)to the cartridge lane (112(b) and 112(c)) at one of two locations. Forexample, attaching the identification bar 112(f) towards the front ofthe system may configure that cartridge lane (112(b) or 112(c)) for RNAassay cartridges; attaching the identification bar 112(f) towards theback of the system may configure that cartridge lane (112(b) or 112(c))for DNA assay cartridges. Alternatively, cartridge lanes (112(b) and112(c)) may be configured without an identification bar 112(f) or withidentification bars 112(f) at both positions in order to designateadditional assay cartridge types. In some embodiments, identificationbars 112(f) can have a square cross-section, however otherconfigurations, including asymmetric cross-sections, are possible. Theremay also be a sensor under each identification bar 112(f) position foreach cartridge lane (112(b) and 112(c)) that can detect the specifiedconfiguration. Each identification bar 112(f) can also include indiciato alert the operator to the configuration.

Although identification bars are described in detail, it is understoodthat embodiments of the invention are not limited to the use ofidentification bars and that any suitable cartridge identificationdevice can be used. For example, instead of identification bars, eachcartridge could have an RF ID tag (or other identification device) thatcould be detected by a sensor in each cartridge lane 112(b), 112(c).Such identification mechanism may be mechanical in nature, or may usesome electrical, optical, or magnetic mode of operation.

As shown in FIG. 4( a)-1 an assay cartridge 200 can be designed to havea keying feature 224, which may be placed at different locations on thevertical web 226 or other suitable locations of the assay cartridge 200to interface with the identification bar 112(f) and designate differentassay cartridge 200 types. When an assay cartridge 200 is placed in acorrectly configured cartridge lane (112(b) and 112(c)) of the CLU 112the identification bar 112(f) enters this keying feature. Failure of anassay cartridge 200 to seat properly within the cartridge lane (112(b)and 112(c))) may alert the operator to the use of an incorrect assaycartridge. Other assay cartridge 200 types may be designated byincorporating a keying feature 224 that includes a wide notch thataccommodates multiple identification bars 112(f) within a cartridge lane(112(b) and 112(c)). Assay cartridges 200 may also be designed without akeying feature, for occupation of cartridge lane (112(b) and 112(c))configured without an identification bar 112(f).

The use of the above-described keying features and identification barhas a number of advantages. Because the keying features andidentification bar are visible to the user, the user cannot make amistake by putting the wrong cartridge in the wrong cartridge lane.Further, if an assay cartridge is placed in the wrong position, then itmay not be possible to close the cover of the CLU. Embodiments of theinvention thus reduce the chance of operator error.

As seen in FIG. 7( a), the CLU presentation lane 113 may be placedadjacent to the onload module 119. An embodiment of the CLU presentationlane 113 is shown in more detail in FIG. 7( c). The presentation lane113 may include a presentation carriage 113(b) that moves along a CLUpresentation rail 113(c), a CLU presentation guide 113(a) that providesaccurate location of the assay cartridge 200 in the X and Z direction,and a CLU presentation vertical support 113(d) that is coupled to andprovides support for the aforementioned structures. The CLU presentationcarriage 113(b) may be driven by a stepper motor and timing belt in amanner similar to that used by the presentation carriage of the samplepresentation unit 110. In one embodiment, the CLU presentation lane 113accepts an assay cartridge 200 from either of the two cartridge lane(112(b) and 112(c)) and then transports the assay cartridge 200 into thesystem for processing. The cartridge presentation lane 113 may be in themotion path of the sample pipettor 700. In such an embodiment, thecartridge presentation lane 113 may include an orifice or gap 111 (shownalso in FIG. 7( a)) in the presentation guide 113(a) through which themillitip pipettor 704 of the sample pipettor 700 can access the assaycartridge 200. Another gap 113(f) may also be present in thepresentation guide 113(a) to allow access to an XYZ transport device.The CLU presentation lane 113 may include multiple interface points withan external device, such as the XYZ gantry 130, in order to addressscheduling needs and reduce contamination issues. In some embodiments,the system may have cartridge presentation lanes arranged at both endsof the onload lanes.

A drive assembly 113(e) may be coupled to the vertical support 113(a).It can be used to drive the cartridge carriage 113(b) along the CLUpresentation lane 113. It may include components such as a drive pulley,a spring tensioner, and a drive belt.

In some embodiments, the presentation lane 113 may be temperaturecontrolled. Temperature control of the presentation lane 113 be may beachieved by the inclusion of thin film heaters, infrared emitters,thermoelectric devices, a flow of heated air through the unit, or othermeans. Such devices may be attached to the CLU presentation verticalsupport 113(d). Alternatively, the CLU presentation guide 113(a) mayinclude one or more skirts that are proximate to the assay cartridge 200and permit incorporation of temperature control devices by similarmeans. The presentation lane 113 may also include a device for measuringthe temperature of the reagent pack. Suitable temperature sensingdevices include infrared temperature sensors.

Embodiments of the invention may include other variations. For example,although two onload lanes are shown in the embodiments that aredescribed above, other embodiments of the invention may include one tothree or more onload lanes for different cartridge types. Further, otherembodiments of the invention may comprise a dedicated bypass lane or aloading position for a “one off” cartridge. For example, if the systemis normally only loaded with DNA cartridges and there is anunanticipated need to run an RNA assay; a single RNA cartridge could beloaded into a bypass onload lane (or other separate, designatedposition) rather than having to unload and re-key one of the onloadlanes. In yet another embodiment, there could be a dedicated STAT (shortturnaround time) position or lane for an assay cartridge designated foruse with a STAT sample. In still another embodiment of the invention,the cartridge loading unit may hold assay cartridges 200 in a radial orcircular arrangement such as, for example, supported by a turntable.

Yet other embodiments of the invention can relate to the use of anonspecific onload lane holding mixed cartridge types, where the systemutilizes a pick-and-place device to select and transfer individualcartridges into the presentation lane. A vision system can also be usedto distinguish different assay cartridge types.

Other functional features may be included in the CLU. For example, itcan be desirable to incorporate a mixing device into the CLU to suspendcartridge contents. For example, an orbital mixer or ultrasonic mixercould be used in some embodiments of the invention.

H. Reagent Storage Unit

FIG. 8( a) shows a top perspective view of a reagent storage unit

FIG. 8( b) shows an enlarged view of the front of a reagent storage unit

FIG. 8( c) shows an interior wall of a reagent storage unit

The reagent storage unit 124, or RSU, may be used as a repository forreagent packs 400 on the system. The reagent storage unit 124 canfacilitate on-system storage of reagent packs 400, advantageouslyimproving the stability of reagents on the system and reducing the needto store reagents in a separate device when the system is not in use.The RSU may have a pressure sensor (not shown) for sensing ambient airpressure.

In one embodiment of the invention, the reagent storage unit 124 has abaseplate 132, a proximal wall 130 of a body disposed on the baseplate132, a distal wall 148 opposite the proximal wall 130, and a cover 128.The baseplate 132, distal wall 148, and the proximal wall 130 may definea cavity. The cover may include a dampening spring to control the rateof opening. The interior surface of the reagent storage unit 124 mayincorporate guide features 136 that align the assay reagent packs oninsertion.

The unit may be temperature controlled in order to maintain theintegrity of the reagents. Different areas of the reagent storage unit124 may be maintained at different temperatures. Temperature control maybe provided by one or more thermal electric units 134 that are inthermal communication with the baseplate 132 of the reagent storage unit124. Other means of providing temperature control include the use ofchannels within the baseplate 132 that conduct fluids, direction ofchilled gases into the interior of the reagent storage unit 124 oragainst a surface in thermal contact with the unit, and positioning amechanical refrigeration unit in thermal contact with the reagentstorage unit 124. Such temperature control devices may furtherincorporate heat exchangers and fans or similar devices in order moreefficiently remove heat from the reagent storage unit 124. Otherfeatures to maintain reagent integrity during storage, such as mixingdevices to keep reagent pack 400 contents mixed and in suspension, maybe incorporated into the reagent storage unit 124. Such mixing devicesinclude rockers, orbital mixers, and ultrasonic devices.

The cover 128 of the reagent storage unit 124 may include one or moreaccess doors 126, as shown in FIG. 8( a). These can be opened in orderto add or remove reagent packs and closed during normal operation. Inone embodiment, the access door 126 of the reagent storage unit 124 isconstructed in one or more sections that are attached to cover 128 by ahinge. Alternatively, the access door 126 may move along a trackincorporated into the reagent storage unit 124. This door 126 serves toreduce contamination, control evaporation, and to help control thetemperature within the reagent storage unit 124. In some embodiments,the access doors 126 can be opaque to protect light sensitive reagents.

As shown in FIG. 8( b) the proximal wall 130 of the reagent storage unit124 may also include one or more status indicators 140 that indicate thecondition of assay reagent packs held within the unit. These statusindicators 140 may indicate the presence or absence of an assay reagentpack at a particular location within the reagent storage unit 124,indicate that an assay reagent pack 400 needs to be replaced, orotherwise provide the user with cues to the operation of the unit. Inone embodiment, the status indicators 140 are color-encoded LEDs;alternative embodiments include but are not limited to incandescentlamps, an LCD display, or other suitable visual indicators. In anotherembodiment, the reagent storage unit 124 may incorporate audible alarmsto indicate the status of reagent packs 400 stored therein. In yetanother embodiment, the reagent storage unit 124 may provide informationto the system controller related to the status of reagent packs 400stored therein. In yet another embodiment, the status indicators 140 maybe replaced with user notifications on the system monitor or on a remotedevice (e.g., a mobile device).

The distal wall 148 may include mechanisms that secure the reagent packswithin the reagent storage unit 124 and means for addressing read/writememory devices incorporated into the reagent packs 400, as shown in FIG.8( c). The interior of the distal wall 148 of the reagent storage unit124 can include one or more latch assemblies 144 for securing thereagent pack 400, which may include a mechanical latch. The RSU latchassembly 144 may be similar in design to the rack clasp 554 of themicrotip storage unit 120 shown in FIG. 13( d). In one embodiment, uponcontact with the reagent pack 400, the latch assembly 144 is biasedagainst it, and pressure is provided by a pliant member such as aspring. The spring may also act as a ground path for other components ofthe reagent storage unit 124, such as a thermal electric unit 134. Thereagent pack 400 can be released from this latch assembly 144 whenpressure is applied to the latching mechanism by the XYZ gantrypipettor, using a disposable microtip 542. In an alternative embodiment,the distal wall may include apertures positioned such that addition of anew reagent pack 400 to a position occupied by a spent reagent packpushes the spent reagent pack through the aperture associated with thatstorage position. In such an embodiment, the spent reagent pack would bedirected to a waste container.

In one embodiment, the distal wall 148 of the reagent storage unit 124may also include a reagent pack reader 146, which includes a device forinterrogating addressable memory units 426 incorporated into the assayreagent packs 400, as shown in FIG. 9( c). Addressable memory units 426may include RFID chips, contact memory devices such as 1-Wire devices,and iButton (registered trademarks of Maxim Integrated Products, Inc. ofSunnyvale, Calif.) devices. These may store information related tospecific lots of reagent, information related to the cartridge to thememory unit is attached, or both. The distal wall 148 of the reagentstorage unit 124 may also include devices for detecting the presence ofan assay reagent pack 400, including but not limited to a Hall effectsensor, an optical sensor, or a gravimetric sensor.

Reduced temperatures within the reagent unit can lead to the formationof condensation on the interior surface of the cover 128, particularlyin humid environments. Since this condensation may be a source ofcontamination should it fall into a reagent pack 400, the reagentstorage unit cover 128 may be in thermal contact with one or moreheating devices. Such heating devices warm the cover 128, advantageouslypreventing the buildup of condensation without overwhelming the capacityof cooling devices that are in thermal contact with the baseplate 132.Suitable heating devices may include resistance heaters, thin filmheaters, and infrared emitters. The interior temperature of the reagentstorage unit 124 may be maintained through the use of one or moretemperature sensors that form part of a temperature feedback loop.

In one embodiment, the reagent storage unit cover 128 also includesholes, piercings, channels, or similar entry means for a pipettingdevice to access the contents of assay reagent packs 400 held within thereagent storage unit 124 without the need for opening the unit andexposing its contents to the environment. Such openings may also beprovided in order for the XYZ gantry to release a latch assembly 144that secures a reagent pack 400 within the RSU 124, as noted above. Insome embodiments, the reagent storage unit cover 128 is protected by aset of actuated doors, which cover the piercings or other entry meanswhen the reagent storage unit 124 is not being accessed.

FIG. 8( d) shows a front perspective view of a reagent storage unitaccording to another embodiment of the invention. FIG. 8( e) shows aportion of a front perspective view of a reagent storage unit accordingto another embodiment of the invention. In FIGS. 8( d) and 8(e), the RSUcover 128, the RSU distal wall 148, the access door 126, the baseplate132, the cold plate 138, the guide feature 136, and the proximal wall130 in the reagent storage unit 124, as well as the reagent pack 400 andthe reagent pack handle 406, are described above, and the descriptionsabove are incorporated herein.

FIG. 8( d) additionally shows an acoustic noise barrier 166 at a frontof the reagent storage unit 124, and alignment pins 164 at a rear of thereagent storage unit 124. The acoustic noise barrier 166 can compriseany suitable sound insulating material (e.g., a noise reducing foam), toreduce the noise generated by internal components (e.g., a fan) of thereagent storage unit 124.

FIG. 8( f) shows a side, perspective, cross-sectional view of a reagentstorage unit. FIG. 8( g) another side, perspective, cross-sectional viewof a reagent storage unit. As shown therein, the reagent storage unit124 can have a heat source at a top region of the reagent storage unit124, and a cold source at a bottom region. As shown in FIG. 8( f), thetop can include a heater 172, which can serve to reduce condensationthat may be a source of contamination. It can use any suitable heatdevice including an electrical heating coil, heating coils with hotfluids passing through them, and one or more thin film heaters Thereagent storage unit 124 can include a tapered floor 170 that serves toguide condensate away from the unit, and can be operatively coupled to afinned heat sink 174 and a fan 180. The fan 180 may be controlled by acontroller (e.g., on a data board 168) that utilizes data provided bysensors to modulate fan speed and thereby minimize noise, and it may becoupled to an intake manifold 186 (shown in FIG. 8( g)), and an exhaustmanifold 188. Such sensors may monitor ambient temperature, ambienthumidity, and internal temperature of the reagent storage unit 124. Aseal 184 can prevent mixing of ingoing and outgoing air. Referring againto FIG. 8( f), a condensate trough 173, a condensate port 176, and acondensate tray 178 may be used to remove condensate from the taperedfloor 170 of the cold plate 130.

In embodiments of the invention, an algorithm can utilize information onambient air pressure, ambient temperature, and heat sink temperature tocontrol fan speed. This can advantageously reduce noise and powerconsumption. The logic for the algorithm may reside in a memory unit(e.g., a memory chip) on a data board 168 in the reagent storage unit124 or remote from it.

FIG. 8( h) shows a perspective, cross-sectional view showing a rearportion of a reagent storage unit. As shown, the previously describedlatch assembly 144 may comprise a latch 144(a), which can be biased intoa forward position by a latch spring 144(b). The latch spring 144(b)could be a flexible strip of metal, a torsion spring, or other biasingelement. FIG. 8( h) also shows a pack pressure sensor 192, as well as anelectrical contact 190. These elements can sense the presence of thereagent pack 400. The electrical contact 190 can also be used to readinformation from a memory element attached to the reagent pack 400.

FIG. 8( h) also shows a first aperture 128(a) and a second aperture128(b) in the cover 128. The first aperture 128(a) is disposed above awell 400(a) of the reagent pack 400. A pipettor (not shown) can access areagent in the reagent well 400(a).

The second aperture 128(b) provides access to one end of the latch144(a), so that a probe (such as a pipette tip) can be inserted into thesecond aperture 128(b) and can provide downward force, thereby causing arear releasing feature 144(a)-2 of the latch 144(a) to move down whilethe front fastener 144(a)-1 of the latch 144(a) pivots up. A pivotportion 144(a)-3 is between the fastener 144(a)-1 and the releasingfeature 144(a)-2. Once this happens, the latch 144(a) disengages fromthe latch pocket 430 (which may be an example of a mating feature) ofthe reagent pack 400. The reagent pack 400 is pushed outward (ejected)and toward the front of the reagent storage unit 124 by the springejection plate 194 secured to a rear wall 149 of the reagent storageunit 124. This advantageously distinguishes the reagent pack 400 to beremoved from the reagent storage unit, simplifying this task for theuser. The sprint ejection plate 194 could be any other suitableresilient member (e.g., a spring).

Thus, one embodiment of the invention is directed to a method comprisingaligning a probe with an aperture in a storage unit. The storage unitcould be a reagent storage unit. Then, the method includes inserting theprobe through an aperture in the storage unit and pushing a latch as theprobe is inserted through the aperture, thereby causing the latch todisengage from a latch pocket of a consumable pack held within thestorage unit. The consumable pack may be a reagent pack or a pack ofpipette tips, etc. Such embodiments advantageously use a probe (e.g., apipette) that may have other uses including pipetting or movingcomponents within the system.

In an alternative embodiment, a latch 144(a) could be pivoted out of thelatch pocket 430 of a reagent pack 400 by applying pressure using alinear actuator. Such linear actuators can include a solenoid, motordrive, hydraulic or pneumatic ram, or other suitable actuator.

In some embodiments, as shown above, the reagent pack further comprisesa second well, and the cover further includes a third aperture, and thethird aperture of the cover aligns over the second well therebyproviding the pipettor access to the second well. The first, the second,and the third apertures are arranged linearly in such embodiments (e.g.,as shown in FIG. 8( h), the apertures in the cover 128 above the reagentwells including reagent well 400(a) and the releasing feature 144(a)-2are aligned in a linear fashion).

FIG. 8( i) shows a portion of a reagent storage unit cover as itinterfaces with a containment feature 197 of a reagent pack 400. Bothsides of the reagent pack 400 may include L-shaped (or other shaped)containment features that can conform to an internal cover wall 128(b).As shown, there can be multiple parallel walls 128(b) expending downwardfrom a major horizontal portion of the cover 128. These features canhelp to ensure that the reagent pack 400 is property situated in itscorresponding slot in the reagent storage unit 124.

As an alternative to reagent storage units that hold the reagent pack ina fixed position, other embodiments include a reagent storage unit inwhich reagent packs are stored in a temperature controlled storage unit,such as a refrigerator, and moved to a reagent pipetting area as needed.In still another embodiment of the invention, the reagent storage unitmay hold reagent packs 400 in a radial or circular arrangement such as,for example, supported by a turntable. In still another embodiment ofthe invention, the reagent storage unit may hold reagent packs 400 in aradial or circular arrangement such as, for example, supported by aturntable. In such an embodiment, the reagent packs 400 may be stored ina rotary carrier that spins on its central axis to present a specificreagent pack to a pipetting device. Alternatively, reagent packs 400 maybe stored in fixed locations and accessed by transfer devices withmultiple degrees of freedom. Transfer devices for such an embodimentinclude an XYZ manipulator or articulated arm with a suitable grippingor support feature.

I. Reagent Pack

FIG. 9( a) shows a top perspective view of a portion of a reagent pack

FIG. 9( b) shows a cutaway view of a reagent pack

FIG. 9( c) shows an exploded view of a reagent pack

FIG. 9( d) shows a barrier lid of a reagent pack

FIG. 9( e) shows an end portion of a reagent pack

The system may store reagents in the form of a reagent pack 400. In someembodiments, as shown in FIG. 9( a), a reagent pack 400 can be amulti-use consumable that contains reagents useful for performing anassay type multiple times. The reagent pack 400 may store sufficientreagents to support the performance of 20 to 100 individual assays of aspecified type. In one embodiment, the reagent pack 400 storessufficient reagent to support the performance of 50 (or more or lessthan this) individual assays of a specified type. The system dedicateseach reagent pack to a single assay type and requires only a singlereagent pack 400, in combination with an assay cartridge 200, to supplyall reagents needed for an assay. In some embodiments, reagent packs 400store reagents used for multiple assay types. Reagents stored in thereagent pack 400 may be stable at ambient temperatures. Alternatively,reagents stored in the reagent pack 400 may use refrigerated storage forstability.

The system design can allocate reagent storage between reagent packs 400and assay cartridges 200, based on assay specificity and storagecondition needs. In some embodiments, reagents stored in assaycartridges 200 can be determined by specimen type. For example, a DNAassay cartridge can store reagents related to DNA extraction andpurification regardless of whether the system uses that assay cartridgeto perform a Chlamydia trachomatis (“CT”) and Neisseria gonorrhoeae(“NG”) assay or a cytomegalovirus (“CMV”) assay. In one embodiment,reagent packs 400 store reagents that are specific for a particularanalyte. In another embodiment, reagent packs 400 store reagents thatrequire refrigerated storage. In yet another embodiment, reagent packs400 store both reagents that are specific for a particular analyte andreagents that require refrigerated storage. Examples include but are notlimited to: (1) a CMV reagent pack storing amplification primersspecific for a CMV assay, (2) a reagent pack storing achromopeptidase orproteinase K enzymes that are used for multiple assay types and requirerefrigerated storage, and (3) a reagent pack storing both (a)amplification primers for a CT and NG assay and (b) achromopeptidase orproteinase K enzymes that are used for multiple assay types. Other typesof reagents may be used in other embodiments of the invention. Materialsmay be transferred between reagent receptacles (408, 414) of a givenreagent pack 400 while it is stored in the reagent storage unit 124. Insome embodiments materials may be transferred between reagentreceptacles (408, 414) of different reagent packs 400 while the reagentpacks are stored in the reagent storage unit 124.

As shown in FIG. 9( a), the reagent pack 400 can include a generallyrectangular elongated body formed to include multiple reagentreceptacles including one or more large reagent receptacles 408, and oneor more relatively smaller reagent receptacles 414, as well as featuresto facilitate handling and automation. The large and small receptacles408, 414 are aligned in a linear array in this embodiment.

In some embodiments, the reagent pack 400 may be manufactured byinjection molding. Alternatively, the reagent pack 400 may bemanufactured by assembling individual reagent receptacles 408, 414. Insuch an embodiment individual reagent receptacles 408, 414 may be joinedusing adhesives, by welding, or by fixing to a framework.

The reagent pack can have a proximal end 450 and a distal end 404 atopposite termini of the elongated body. The orientation of the reagentreceptacles defines the top and bottom of the reagent pack; reagentreceptacles are open at the top and closed on the bottom and sides. Thereagent pack 400 may be opaque to protect photosensitive reagents fromlight. In one embodiment, the reagent pack 400 is made from acarbon-filled plastic, which may be conductive or have antistaticproperties.

In some embodiments, the reagent receptacles (408, 414) align in asingle row (or be in a linear array) along the reagent pack long axis.This advantageously provides for compact storage, and additionallyallows heat transfer surfaces to flank two sides of each reagentreceptacle during storage. This two-sided proximity helps maintainreagents at the desired storage temperature, improving reagent stabilityand helping to assure reagent quality. Reagent receptacles 408, 414 canbe open top containers of generally rectangular cross-section, orientedparallel to the major axis of the reagent pack 400. This arrangementproduces good thermal contact with fixed heat transfer surfaces when auser slides the reagent packs into the reagent storage unit 124.

Reagent receptacles 408, 414 may be defined by relatively thin walls toallow for rapid heat exchange. A vertical wall 447 may separate adjacentreagent receptacles 408, 414. In one embodiment, individual reagentreceptacles 408, 414 do not share walls with other reagent receptacles408, 414. Separate walls advantageously prevent fluid creep betweenadjacent reagent receptacles 408, 414 reducing the possibility ofreagent contamination. The reagent receptacle walls may extend below thebottoms to form standing features 444 that terminate at a common heightand support the reagent pack on flat working surfaces.

Reagent receptacles 408, 414 may taper towards the bottom for easiermolding. As shown in FIG. 9( b), the bottom of each reagent receptacle408, 414 may also angle downwards centrally to minimize dead volumeduring pipetting. In some embodiments, the bottom portion 446 of eachreceptacle has an inverse pyramidal configuration.

A reagent pack 400 according to an embodiment of the invention mayaccommodate sufficient volumes of reagents for multiple instances of anassay. In some embodiments, each reagent pack 400 includes reagents forabout 20 to about 100 instances of an assay and in some cases about 50instances. In some embodiments a reagent pack 400 may supplied withempty or partially filled reagent receptacles (408, 414), to whichreagents are subsequently transferred from bulk containers, such asbottles. Individual reagent receptacles may differ in dimension toaccommodate the requirements of an assay type. Factors that candetermine the size of a reagent receptacle include the number of usesdesired for the reagent pack type, concentration dependent stabilityissues with reagent components, and the need to minimize the volume ofthe final reaction mixture. As noted above, in some embodiments, eachreagent pack can include a large reagent receptacle 408 and a pluralityof small reagent receptacles 414. In one embodiment a reagent pack 400has six or more small reagent receptacles 414. Each reagent receptacle408, 414 can be large enough to accommodate a microtip 542 used toremove a volume of reagent for use in an assay. In a preferredembodiment, large reagent receptacles 408 have the capacity to storeabout 3.0 mL of fluid and small reagent receptacles 414 have thecapacity to store about 1.2 mL of fluid. Each reagent receptacle 408,414 can includes additional capacity to maintain at least a 7 mmheadspace 452 between the liquid surface of a reagent 448 and a barrierlid 418 that overlies the reagent receptacle 408, 414 when filled withthe reagent 448. The headspace 452 (which may be filled with air) mayserve to insulate the stored reagent from heat applied to the top of thereagent pack 400 when held within the reagent storage unit 124.

As shown in FIGS. 9( a) and 9(c), the reagent pack 400 may includefeatures to facilitate handling and automation, including a containmentsection 412 (which includes the receptacles 408, 414), a gripping handle406, a barrier lid 418, a storage cover 416, an electronic memory 426,labeling, features to engage the reagent storage unit 124, and selectedreagents. In some embodiments, the body of the reagent back 400 may bemade by a manufacturing process that includes injection molding.

A reagent pack 400 according to an embodiment of the invention mayinclude a containment section 412. The containment section 412 may bedefined at least in part by containment walls 422 defining parts of thesides of the reagent pack 400. The containment walls 422 may also beadjacent to or coincide with the distal end 404 and the proximal end450, and may surround the upper openings of the reagent receptacles 408,414. Further, a containment floor 410 may also connect the containmentwall 422 to the openings of each reagent receptacle. In one embodiment,the containment floor 410 is a horizontal web that is contiguous withboth the openings of the reagent receptacles 408, 414 and thecontainment walls 422. The containment section 412 can serve to preventcontamination through containment of drips or spills of liquids that mayoccur during processing or handling. A centrally disposed vertical webmay connect reagent receptacle walls below the containment floor to addrigidity. The walls that define each reagent receptacle 408, 414 mayextend vertically as rims above the containment floor 410 to prevent theincursion of fluids dripped or spilled in the containment region intothe reagent receptacles 408, 414. In some embodiments, these rims mayalso be energy directors 428 (see FIGS. 9( b) and 9(e)) used during theattachment of closures, such as the barrier lid 418, to one or morereagent receptacles 408, 414. These rims may also support leak testingof the sealed reagent receptacles 408, 414 during reagent pack 400manufacturing.

The barrier lid 418 may individually seal the reagent receptacles toprotect the reagents from environmental factors and to prevent reagentcross-contamination. The barrier lid 418 can be a single part spanningall of the reagent receptacle openings 408, 414. Alternatively, thebarrier lid 418 may be a series of individual sealing members that coverindividual reagent receptacle 408, 414 openings. In another embodiment,the barrier lid 418 may be a combination of a single part that spansmultiple reagent receptacle 408, 414 openings and individual sealingmembers that cover individual reagent receptacle 408, 414 openings or anindividual sealing member that covers a single reagent receptacle 408,414. In yet another embodiment, the barrier lid 418 may be a multilayercomposite of polymer foils and a formed polymer support. The polymersupport may confer rigidity to the barrier lid 418, may provide featuresto align the barrier lid 418 with the reagent receptacles 408, 414, andmay provide further isolation features, such as raised lips 418(b)around each reagent receptacle location in the barrier lid, as shown inFIG. 9( d). Such raised lips 418(b) can help keep the user's fingersfrom touching and contaminating the portion of the barrier lid 418immediately atop the reagent receptacles 408, 414. In some embodiments,the barrier lid 418 includes at least one compliant elastomericcomponent that permits the barrier lid 418 to at least partially resealafter piercing. The compliant elastomeric component may be in the formof a strip of preformed caps 418(a) joined by gates and runners (seeFIG. 9( d)).

FIG. 9( d) shows that the barrier lid 418 can include an orientation tab418(c) projecting asymmetrically from one end prevent the lid from beingplaced on the reagent pack in the wrong orientation duringmanufacturing. In one embodiment, the manufacturing process isover-molding of the formed polymer support to the elastomeric component.Suitable materials for the polymer support include polypropylene, suchas natural PURELL X50109 manufactured by LyondellBasell Industries ofRotterdam, The Netherlands. Other suitable materials for the polymersupport include, but are not limited to, polyethylene, nylon,polystyrene, and other polymers with suitable stiffness. Suitablematerials for the elastomeric component may be a thermoplastic elastomersuch as DYNAFLEX® G7930, GLS grade 67930-1001-00 manufactured by GLSCorporation of McHenry, Ill. Other suitable materials for theelastomeric component of the barrier lid 418 include, but are notlimited to, silicone elastomer, latex, and natural rubber.

In operation, a pipette tip (not shown) pierces a barrier lid 418 (e.g.,a preformed cap 418(a) of the barrier lid 418) to access a reagentreceptacle's 408, 414 contents. The manufacturing process may pre-scorethe barrier lid 418 so that tearing during piercing occurs inpredictable locations. In some embodiments, the manufacturing processlaser welds the barrier lid 418 to the rims of each reagent well 408,414. Alternatively, the manufacturing process may use other suitableprocesses attachment methods to fix the barrier lid 418 to the reagentreceptacles 408, 414, including but not limited to heat sealing,ultrasonic welding, induction welding, or adhesive bonding.

Reagents packs may include a storage cover 416 designed to protectreagent pack contents during shipping, off-system storage, or handling,as shown in FIG. 9( c). The storage cover 416 may be a single use“tear-off” cover loosely affixed to the upper surface of the containmentwalls 422. In some embodiments, the storage cover is a replaceable coverthat is held in place by friction or by an interference “snap fit” tothe containment walls 422. This advantageously allows a user to replacethe storage cover if the reagent pack 400 is removed from the system.The storage cover 416 may include identifying or instructional labeling.

FIG. 9( a) additionally shows that the gripping handle 406 may extendfrom the proximal end 450 of the reagent pack 400 to simplify insertionand removal from the system. Placement of the gripping handle 406 at oneend advantageously allows a user to slide the reagent pack 400 into thereagent storage unit 124 through a relatively small opening, reducingtemperature fluctuations in the reagent storage unit 124 duringinsertion. Further, the end placement helps keep user hands, a possiblesource of nucleic acid contamination, distant from the reagents. Thegripping handle may include an extension along the reagent pack axiswith a recess along the lower surface to serve as a finger hold. In oneembodiment, this extension is hollow, which advantageously reduces theweight of the reagent pack 400. The design of the gripping handle 406,coupled with the low weight of the reagent pack 400, permits the user tosecurely grip the reagent pack 400. The gripping handle 406 may includea label surface that remains visible when the reagent pack 400 isinstalled in the reagent storage unit 124. This label location permits auser to identify individual reagent packs by simple inspection withoutdisrupting system operation.

In some embodiments, an isolation portion 420, shown in FIG. 9( c),further separates the gripping handle from reagent receptacles (408,414) within the reagent pack 400. The isolation portion 420 may be anextended hollow segment with a top wall and parallel side walls, withthe side walls arranged parallel to the axis of the reagent pack. Theisolation portion 420 can serve to separate the gripping handle 406 fromthe reagent receptacles (408, 414) to reduce the likelihood of reagentcontamination from user handling. The isolation portion may be from 0.5inches to 1.5 inches in length. In one embodiment, the isolation portionis about 1 inch in length. The isolation portion 420 may also serve tostabilize the reagent pack 400 when it is placed on a flat surface. Theisolation portion may be from 0.5 inches to 1.5 inches in length. In oneembodiment the isolation portion is about 1 inch in length. Theisolation portion 420 may also serve to stabilize the reagent pack 400when it is placed on a flat surface. A secondary purpose may be toprovide surfaces to support reagent pack labeling.

The reagent pack 400 may also include electronic memory 426 to storeinformation related to the reagent pack 400 and to transfer informationabout the reagent pack 400 to and from the system, as shown in FIG. 9(c). The electronic memory 426 may communicate by electrical contact orwirelessly. In some embodiments, the electronic memory 426 is a contactmemory device utilizing the 1-Wire® protocol manufactured by MaximIntegrated Products, Inc. of Sunnyvale, Calif. In other embodiments, theelectronic memory 426 may be an RFID device, an iButton (registeredtrademark of Maxim Integrated Products, Inc. of Sunnyvale, Calif.)device, or another electronic memory device of suitable dimensions. Theelectronic memory may be mounted anywhere on the reagent pack. In oneembodiment, the electronic memory 426 is affixed to a locating feature432, shown in FIG. 9( e) near the distal end 404 of the reagent pack400. Upon loading into the reagent storage unit 124, the recess may bedisposed proximate to a reagent pack reader 146 (FIG. 8( c)) thatprovides power and information. The memory device 426 may includeinformation entered during reagent pack 400 manufacturing andinformation transferred during use. Information stored in the memorydevice 426 entered during manufacturing may include: assay type, reagentcartridge serial number, lot number, and reagent expiration, andinformation related to the stability of the contents of the reagent packonce it has been accessed by the system. Information entered duringmanufacturing may also be encoded in a one dimensional barcode, a twodimensional barcode, or through similar labeling. Informationtransferred during use may include: the date that the reagent pack wasfirst loaded onto the system, the amount of time the reagent pack hasbeen stored on the system, the number of tests run from the reagentpack, and the number of tests remaining in the reagent pack, and ahistory of which individual systems that the reagent pack has beenloaded onto. In some embodiments, the system writes new information tothe electronic memory 426 after each access of the reagent pack 400 andreads information whenever a user loads a reagent pack.

FIG. 9( e) shows that the reagent pack 400 may include features toengage the reagent storage unit 124 including tapered lead-in features438 to guide the reagent pack during insertion, a pack shoulder 440 tosupport the reagent pack within the reagent storage unit 124, a latchpocket 430 to lock the reagent pack into the reagent storage unit 124, aspring engager 434 to help eject the reagent pack once the systemreleases the reagent pack 400 from the reagent storage unit 124, and asensor flag 466 to indicate the presence of a reagent pack 400 in areagent slot.

Lead-in features 438 may extend from the side walls of the reagentreceptacle closest to the distal end of the reagent pack 404. In oneembodiment, the lead-in features 438 are extensions of the side wallsthat angle toward the midline of the reagent pack, forming a taper thataids the user in centering the reagent pack during insertion into thereagent storage unit 124 reagent storage unit 124.

The containment floor 410 of the reagent pack may extend beyond the sidecontainment walls 422 as a pack shoulder 440. In some embodiments, thepack shoulder 440 is a controlled surface. The pack shoulder 440 mayextend laterally approximately 1-2 mm from either side of thecontainment walls 422 and can serve to locate the reagent pack 400vertically within the reagent storage unit 124 reagent storage unit 124.The lower surface of the pack shoulder 440 may support the reagent pack440 on the RSU cold plate 138 in the reagent storage unit 124 (see FIG.8( b)). This advantageously reduces the effect of tolerance stack-up bylocating the reagent pack 400 with respect to the RSU cold plate 138based on a controlled surface. The upper surface of the pack shoulder440 secures the reagent pack 400 during pipetting operations, when thecompliant portion of the barrier lid 418 may grip an ascending microtip542. An end of the pack shoulder 440 may also include tapered lead-infeatures.

As described in more detail above, the system may secure reagent packs400 within the reagent storage unit 124 using a spring-loaded latchassembly 144 (see FIG. 8( c)). The reagent pack 400 can include a matingfeature, such as a latch pocket 430 that is complementary to a latchingportion of the RSU latch assembly 144. As shown in FIG. 9( e) the latchpocket 430 may be an open rectangular cavity near the distal end 404 ofthe reagent pack 400. In one embodiment, a portion of the containmentwall 422 surrounds the latch pocket; a front portion of the containmentwall joined to extended side portions of the containment wall can definea rectangular opening perpendicular to the axis of the reagent pack,defining a latch pocket 430 that is complementary to the latchingportion of the RSU latch assembly 144. The latch pocket 430 may becovered by the storage cover 416 prior to use, preventing the user fromsuccessfully loading a reagent pack 400 onto the system without firstremoving the storage cover 416.

As described in more detail above, the system may eject released reagentpacks. FIG. 9( e) shows an extension of the vertical wall 424 at thedistal end of the reagent pack 404 that can act as a spring engager 434which interacts with an ejection spring. In one embodiment, the springengager 434 is located proximate to the lower surface of the reagentpack near the midline. The upper portion of this extension of thevertical wall may also incorporate a sensor flag 46 that interacts witha reagent pack sensor within the reagent storage unit 124 to indicatethe presence of a reagent pack within the reagent storage unit 124.

There can also be a number of other alternative embodiments of theinvention. For example, common reagents used in all assays or sampleprocesses could be held outside of the reagent packs in bulk bottles, orreagent packs could be single use.

J. Processing Lanes

FIG. 10( a) shows a perspective view of a processing lane with anengaged assay cartridge.

FIG. 10( b) shows a side view of a processing lane with an engaged assaycartridge.

FIG. 10( c) shows a perspective view of a processing lane that hasthermal control with an engaged assay cartridge.

FIGS. 10( d) and 10(e) show different perspective views of an embodimentof a processing lane heater.

FIG. 11 shows a side, cross-sectional view of a processing lane of aprocessing lane heater according to an alternative embodiment of theinvention.

The assay cartridges 200 described above are processed by the system inone or more processing areas, which incorporate mechanisms forperforming specific steps necessary for processing a patient sample.Such mechanisms may include fluid transfer devices suited to a volume ofabout 1 mL, or fluid transfer devices suited to a volume of 100 μL to200 μL, or even down to 10 μL or less, temperature control devices,magnetic devices, and devices for performing other necessary functions.A processing area may include one or more of these devices. Theseprocessing areas may include one or more lanes that process the assaycartridge 200 in a linear fashion. In some embodiments lanes thatprocess the assay cartridge 200 may be arranged in a radial or circularfashion. In other embodiments, processing areas may include rotatingcarousels, areas where the assay cartridge is immobile and accessed byprocessing mechanisms on a gantry system or articulated arm, or otherconfigurations that permit access to the assay cartridge by processingmechanisms in a controlled manner.

Referring again to FIG. 1( b), FIG. 1( b) shows an embodiment of thesystem that includes a number of processing lanes 116 for processingassay cartridges 200. The system may include a first, second, third,etc. processing lanes configured to process a sample in an assaycartridge 200. It can also include a transfer shuttle 50, which movesassay cartridges 200 between the processing lanes 116.

In some embodiments, a controller 94 directs operations of theprocessing lanes 116 and the transfer shuttle 50. In one embodiment, thecontroller can store and execute one or more protocols for directingassay cartridges 200 through a series of specified processing lanes 116in a specified order using the transfer shuttle 50. For example, thecontroller 94 may be configured to execute a first protocol and a secondprotocol. In one embodiment, the controller 94, in executing the firstprotocol, directs the transfer shuttle 50 to move an assay cartridge 200from a first processing lane (e.g., a cartridge loading lane) to asecond processing lane (e.g., a heating lane). In executing the secondprotocol, the controller may direct the transfer shuttle 50 to move anassay cartridge 200 from the first processing lane (e.g., the cartridgeloading lane) to a third processing lane (e.g., a wash lane) withoutmoving the assay cartridge to the second processing lane (e.g., theheating lane). Thus, in embodiments of the invention, cartridges can betransferred between adjacent or non-adjacent lanes in any suitablemanner. Non-limiting examples of first, second, and third processinglanes can be selected from the group consisting of a heating laneconfigured to warm an assay cartridge, an amplification preparationlane, a temperature stabilization heating lane configured to maintainthe temperature of an assay cartridge, an elution lane, and a wash lane.

In other embodiments of the invention, the system includes a firstprocessing lane configured to perform operations on a sample in an assaycartridge 200, a transfer shuttle 50 to move assay cartridges into andout from the first processing lane, and a controller 771 to directoperation of the system. The controller 94 may be configured to controloperations in the first processing lane and the transfer shuttle 50.Such a controller may be configured to execute a first protocol and asecond protocol. The controller, in executing the first protocol,directs the transfer shuttle to move a first assay cartridge 200 intothe first processing lane. After a fixed interval, the controllerdirects the transfer shuttle 50 to move the first assay cartridge 200out of the first processing lane. Within the fixed interval, thecontroller directs the first processing lane to execute a first sequenceof operations. The controller, in executing the second protocol, directsthe transfer shuttle 50 to move a second assay cartridge 200 into thefirst processing lane. After the fixed interval, the controller 94directs the transfer shuttle 50 to move the second assay cartridge outof the first processing lane and directs the first processing lane toexecute a second sequence of operations. This sequence of operations ofthe first protocol may be different from the sequence of operations ofthe second protocol.

Flexibility in both the routing of assay cartridges 200 betweenindividual processing lanes 116 and in the operations performed within agiven processing lane gives the system a high degree of operationaladaptability.

The system can include processing lanes 116 that perform the operationalsteps needed for nucleic acid extraction and purification from abiological or patient sample. Each processing lane 116 can accommodatean assay cartridge 200. When the system uses a linearly arranged assaycartridge 200 each processing lane may extend linearly relative to thelong axis of the assay cartridge. Such processing lanes 116 may mirrorthe dimensions of the assay cartridge 200, reducing the need to orientthe assay cartridge and permitting the system to package multipleprocessing lanes in a space-efficient parallel manner. In someembodiments, the system includes processing lanes that are physicallyarranged in an order approximating their order of use in at least someprotocols. This advantageously minimizes the distance and time thesystem needs to transfer assay cartridges between processing lanes.Alternatively, the system may include processing lanes with similarfunctions grouped together. This advantageously minimizes the time spentperforming repetitive functions, such as, for example, washing.

As show in FIG. 1( b) the system may include different types ofprocessing lanes that support functions appropriate to differentprocessing steps. In some embodiments, the system includes multiplereplicates of some lane types, allowing processing of multiple assaycartridges 200 in parallel. Examples of processing lane types include acartridge loading lane 116(f), a transfer lane 50, a heated temperaturestabilization lane 116(j), a wash lane 116(a) and 116(b), an elutionlane 116(e), an amplification preparation lane 116(g), and a waste lane116(c). In some embodiments, the system includes 13 processing lanes inthe following sequence:

LANE POSITION LANE TYPE 1 AMPLIFICATION PREPARATION LANE 2 CARTRIDGELOADING LANE 3 ELUTION LANE 4 WASTE LANE 5 HEATED TEMPERATURESTABILIZATION LANE 6 AMBIENT TEMPERATURE STABILIZATION LANE 7 AMBIENTTEMPERATURE STABILIZATION LANE 8 WASH LANE 9 WASH LANE 10 WASH LANE 11WASH LANE 12 WASH LANE 13 WASH LANEThe first lane position can be near the center of the instrument, withsuccessive lanes numbered toward the right side of the system as viewedfrom the front. Successive lane positions may be disposed adjacent thepreceding lane position. Alternatively, the system may incorporate oneor more processing lanes that individually incorporate all of theprocessing tools needed to perform every processing step.

Another embodiment of a system with different types of processing lanesconfigured to perform different steps is shown in FIG. 20( h). In thisembodiment, the system includes a cartridge warming lane, which servesto rapidly bring the temperature of the cartridge and its contents tothe temperature required for consistent sample processing. In such anembodiment, the system may have 10 processing lanes, some of which arereplicates, in the following sequence:

LANE POSITION LANE TYPE 1 AMPLIFICATION PREPARATION LANE 2 CARTRIDGELOADING LANE 3 ELUTION LANE 4 CARTRIDGE WARMING LANE 5 WASH LANE (SMALLMAGNET) 6 WASTE LANE 7 WASH LANE (LARGE MAGNET) 8 WASH LANE (LARGEMAGNET) 9 WASH LANE (LARGE MAGNET) 10 TEMPERATURE STABILIZATION LANEWITH PIPETTE PUMPEmbodiments of the invention may use one or more of the above describedlanes, in any suitable combination.

Referring to FIGS. 10( a)-10(c), a processing lane may include a lanesupport 834 to retain processing lane components, a cartridge guide 800to support and guide an assay cartridge 200, a cartridge carriage 816 tomove an assay cartridge 200 along a lane motion path within theprocessing lane, and a transfer position to interact with the transportshuttle 898 (shown in FIG. 14( e)).

The lane support 834 (see FIG. 20( j)) provides attachment points andholds processing lane components in relationship to one another. In someembodiments, the lane support forms a vertical wall disposed generallyparallel to the axis of an assay cartridge 200 in the processing lane116. The configuration of the lane support 834 may be different indifferent processing lanes 116, conforming to the shape of otherprocessing lane components. The lane support 834 may include mountinglocations for at least some of these components.

The cartridge guide 800 supports an assay cartridge 200 while in aprocessing lane. Its purpose can be to retain the assay cartridge 200during movement. It may also serve to consistently locate the assaycartridge 200 for interaction with processing tools. In someembodiments, the cartridge guide 800 supports a controlled surface thatis part of the assay cartridge 200. In one embodiment, the controlledsurface of the assay cartridge 200 is the bottom surface of thehorizontal web 228 as discussed above. The cartridge guide 800 maysupport the assay cartridge 200 by providing a running surface within aguide channel 862 (see FIG. 10( c)), such a guide channel having across-section that is approximately complementary to the cross-sectionof at least portion of the assay cartridge 200.

In some embodiments, the cross-section of the guide channel 862 isslightly larger than the nominal size of an assay cartridge 200 in orderto reduce friction, prevent jamming, or both. In some embodiments, theguide channel 862 of the cartridge guide 800 is the approximate shape ofan inverted “U”, fixed to the lane support with the open portion of theU facing downwards. Such an inverted U-shape includes a closed top wall,closed side walls depending at about right angles from the top wall, andan open bottom wall connected at about a right angle to the side walls.The open bottom wall may include two horizontal wall segments separatedby a gap, with each segment connected to one of the side walls. This gapforms a channel opening. The various assay cartridge compartments andits vertical web may project through the channel opening.

FIG. 14( f) shows the inside of a shuttle channel 892 and the featurestherein may be similar to those in the guide channel 862. The upperaspect of the bottom wall forms a running surface. The assay cartridgerides upon the running surface, which may support the assay cartridgehorizontal web 228 on one side and the bottom surface of a cartridgeflange 906 on the other side. Since the running surface supports assaycartridge features that may be at two different heights, the twohorizontal wall segments may also be at different heights.

As shown in FIG. 14( f), an indexing wall 893 can be placed below thetop rim of the assay cartridge 200 to minimize contamination by fluidtransfer. The cartridge guide 800 can cover the assay cartridge whereverpossible to minimize contamination. The cartridge guide 800 can have asecondary anti-rotation feature 891 to prevent upwards rotation of theassay cartridge during pipetting operation.

In some embodiments, the cartridge guide 800 includes a retention recessformed within the interior of the U-shaped guide channel 862 as shown inFIG. 10( c). An external view of the cartridge guide 800 is also shownin FIG. 14( e). The retention recess extends along one wall of theU-shaped channel and is roughly complementary in shape to the cartridgeflange 906. The retention recess can act to constrain vertical movementof the assay cartridge 200 during pipetting operations. As discussedabove, such vertical movement may occur due to friction between apipette tip and a barrier film 205; such movement adversely affects theaccuracy of pipetting operations and may lead to spillage withsubsequent contamination of the system.

The cartridge guide 800 may extend along less than the entire motionpath of a processing lane. In a preferred embodiment, the cartridgeguide 800 does not reach into the transfer position. In otherembodiments, such as the waste lane shown in FIG. 14( a), the cartridgeguide does not extend into other operative locations. The transfershuttle 50 may perform the cartridge guide 800 function when an assaycartridge 200 is in the transfer position as described in more detailbelow. The cartridge guide 800 may terminate adjacent certain operativelocations, such as lane heaters 840 (FIG. 10( c)) and 1104 (FIG. 11),where intimate contact between a portion of the assay cartridge 200 andthe operative location is desirable for operation. The extended lengthof the linear-style assay cartridge allows the cartridge guide 800 tosupport the assay cartridge 200 when only a portion of the assaycartridge is engaged within the cartridge guide.

The cartridge guide 800 may include index springs to press the assaycartridge 200 against an internal aspect of one of the side walls of theguide channel 862 in order to better control lateral position of theassay cartridge. Index springs may be strips of a relatively stiff butelastic material, such as spring steel, mounted to a cartridge guide 862side wall. In some embodiments, the index springs mount in openingswithin the cartridge guide 862 side wall.

Any of the walls of the guide channel 800 may include openings orpiercings in one or more locations. In some embodiments openings in theguide channel top wall, give processing tools access to assay cartridge200 compartments. Other openings, such as those described above forindex spring mounting, may serve other functions.

A cartridge pusher (which may be an example of a loading transport) maybe used to position an assay cartridge in any of several operativepositions within a processing lane. The cartridge pusher can include acartridge carriage 816 to engage the assay cartridge 200, a carriagetrack 818 to guide the motion of the cartridge carriage, and a carriagedrive (not shown) to move the cartridge carriage along the carriagetrack.

In one embodiment, the cartridge carriage 816 engages a controlledsurface of an assay cartridge 200 to move the assay cartridge within thecartridge guide 800. The cartridge carriage 816 may also unload theassay cartridge from the transfer shuttle 898 (see FIG. 14( d)), andreturn it. In some embodiments, the controlled surface utilized by thecartridge carriage 816 is a vertically disposed edge of the vertical web226 at the distal end of the assay cartridge 200. A support tab feature218 may be provided on the distal end of the assay cartridge, dependingfrom the assay cartridge a small distance distal to the aforementionedcontrolled surface and thereby defining a gap. The cartridge carriage816 can include a propelling feature 304 (see FIGS. 4( d) and 10(b))that fits within this gap. In this configuration, movement of thecartridge carriage 816 toward the proximal end of the assay cartridge200 drives the propelling feature 304 against the controlled surface.Alternatively, movement of the cartridge carriage 816 away from theproximal end of the assay cartridge 200 drives against the propellingfeature 304 against the proximal aspect of the support tab 218.

In some embodiments, the cartridge carriage 816 positions an assaycartridge 200 at an operative location by driving from a singledirection, by driving the propelling feature 304 against the controlledsurface. This has the benefit of compensating for backlash in the lanemotion path and of reducing the effect of tolerance stack up in theassay cartridge; improving the system's ability to position the assaycartridge 200 within a processing lane 116 consistently.

In some embodiments, the cartridge carriage 816 may engage an assaycartridge 200 using support tabs 218 near both ends. In otherembodiments, the cartridge carriage 816 may engage an assay cartridge200 using a support tab 218 located near only one end. This embodimentadvantageously permits the use of processing lanes 116 that includetools which operate on the external surface of the assay cartridge 200.Such an arrangement can minimize interference between processing lanetools and the cartridge carriage 816. For example, a waste lane 116(c)or a processing lane incorporating a lane heater 116(j) may engage anassay cartridge from only one end.

The cartridge carriage 816 may connect to the carriage track 818 througha moving connection such as a track bearing. In some embodiments, thecartridge carriage 816 includes a magnetically responsive strike plate814 at its proximal end to couple to a magnet trolley 808, as describedin greater detail below. In at least some processing lanes, thecartridge carriage 816 may include a microtip holder to store one ormore microtips 542. The microtip holder can be a shelf that extends fromthe cartridge carriage 816, and includes at least one microtip holdingfeature. In some embodiments, this microtip holding feature is a hole orpiercing through the shelf. The microtip holder may be disposed on thelane motion path so that the cartridge pusher may position a microtip542 (see FIG. 13( f)) under a pipettor in a processing lane. Towardsthat end the microtip holder may located near the distal terminus of thecartridge carriage 816. Alternatively, microtip holding features may beplaced at other positions within a processing lane in which they areaccessible by a suitable pipettor. Such locations include but are notlimited to the cartridge guide 800 and portions of the lane support 834.

The cartridge carriage 816 can also serve as a grounding plane toimprove the accuracy of a liquid sensor. Portions of the cartridgecarriage 816 may be extended to come into close proximity to the wellsof the assay cartridge 200. In embodiments of the invention, liquidsensors can be capacitance based; in such embodiments, bringing a metalobject close to the bottom of a liquid filled well may provide a greaterchange in capacitance that would be observed with liquid alone. Asensing circuit that can include liquid sensing capability is describedin further detail below.

The cartridge carriage 816 may be disposed beneath the cartridge guide800 to engage and drive from the underside of an assay cartridge 200.This arrangement facilitates processing of the assay cartridge 200 usingprocessing tools located above the cartridge. The cartridge guide 800and cartridge carriage 816 both need access to the assay cartridge.While some embodiments include a cartridge guide 800 that is generallydisposed above an assay cartridge 200 and a cartridge carriage 816 thatis disposed below an assay cartridge, this is merely one of a number ofarrangements that may accomplish a similar result. In alternativeembodiments, the system may include a cartridge guide 800 that isdisposed below an assay cartridge 200 and a cartridge carriage 816 thatis disposed above the assay cartridge, a cartridge guide and a cartridgecarriage that oppose each other on either side of an assay cartridge, acartridge guide and a cartridge carriage in an intercalated arrangement,or some combination of these. An arrangement in which the cartridgecarriage 816 is disposed beneath the cartridge guide 800 to engage anddrive an assay cartridge 200 from the underside advantageously limitsthe width of processing lanes 116, subsequently decreasing the distancebetween processing lanes and decreasing the size of an assembly ofprocessing lanes. In arrangements where a large number of processinglanes 116 are present in response to a need for high system throughput,for example, a small decrease in processing lane width can produce aconsiderable reduction in system size. Further, since some processingtools, such as pipettors, require access to the assay cartridge 200 fromabove the disposition of the cartridge carriage 816 beneath thecartridge guide 800 avoids potential interference with processing tools.

In some embodiments, in at least some processing lanes 116, the assaycartridge does not rest fully on the cartridge carriage 816 duringmovement. In such embodiments, the cartridge guide 800 supports theassay cartridge 200 and the cartridge carriage 816 provides motive forceto move it along the motion path. Such an arrangement can simplifyrelease of the assay cartridge 200 from a processing lane configured inthis fashion, for example, for transfer to a waste container followinguse.

A carriage track 818 may be used to guide the motion of the cartridgecarriage 816 and, in some processing lanes, may guide motion of othercomponents such as magnet trolleys 808. In some embodiments, thecarriage track 818 attaches to the lane support 834, oriented parallelto the direction of and extending along at least a portion of the motionpath within the processing lane. The carriage track 818 can link tomoving components such as the cartridge carriage 816 throughcomplementary bearings. In some embodiments, the carriage track 816 is alinear guide rail and the bearings may be caged ball bearing blocks,caged roller bearing blocks, or equivalent devices.

The carriage drive may move the cartridge carriage 816 along thecarriage track 818 by any of a number of drive methods such as a leadscrew and nut, a linear motor, or a pneumatic actuator. In someembodiments, the system uses a drive motor 801 attached to the lanesupport 834 near one end of the carriage track 818 and coupled to adrive pulley. An idler pulley 810 may be attached to the lane support834 near the opposing end of the carriage track 818, by an attachmentthat allows adjustment of the separation distance between idler pulley810 and drive pulley. In such an embodiment, a timing belt 868 mayconnect the drive pulley to the idler pulley and connect to thecartridge carriage 864 via a coupling device 864. Rotation of the motor800 drives the timing belt 868, resulting in movement the cartridgecarriage 816 along the carriage track 818.

Specific types of processing lanes, including transfer lanes 116(h),heating lanes 116(j), and wash lanes 116(b) may include a millitippipettor assembly 704. This serves to transfer fluids among compartmentsof the assay cartridge 200 while in the processing lane. This millitippipettor assembly 704 can include a millitip pipettor that is similar tothe millitip pipettor used for transferring samples, as described above.The millitip pipettor assembly may include a liquid sensor, a pressuresensor for sensing pressure within the millitip pipettor, or both typesof sensors. In some embodiments, the millitip pipettor assembly 704 isdisposed above the cartridge guide 800 at a fixed position along thelane motion path. Thus, in some embodiments of the invention, thecartridge guide can be positioned to align an assay cartridge with afirst pipettor such as a millitip pipettor (or alternatively oradditionally, a second pipettor such as a microtip pipettor). The guidechannel 862 top wall may include a piercing at the fixed position toallow the millitip pipettor to access the assay cartridge.Alternatively, the guide channel may be discontinuous, having a gap at afixed position to allow the millitip pipettor access to the assaycartridge 200. Other components of the millitip pipettor assembly 704may include a lane elevator 832 that serves to raise and lower themillitip pipettor with respect to the cartridge guide 800, a millitipmandrel to engage a millitip 220 from the assay cartridge, a millitipaspirator to drive pipetting action, a millitip ejector to disengage amillitip 220 from a mandrel after use, a liquid sensor 702 to detectfluids, millitips, and alignment features. A description of each ofthese other components of a millitip pipettor assembly is provided inmore detail below.

Some processing lanes 116 may include a microtip pipettor assembly totransfer fluids among compartments of the assay cartridge in theprocessing lane. The microtip pipettor assembly may include a liquidsensor, a pressure sensor for sensing pressure within the microtippipettor, or both types of sensors. In some embodiments, the microtippipettor assembly is substantially similar to the millitip pipettorassembly 704 and disposed in the same fashion. However, the microtippipettor assembly includes a microtip pipettor 1142 similar to thatutilized on the XYZ transport device 1100 described below. Features ofthe microtip pipettor may be substantially similar to those of themillitip pipettor 704 used for aspiration of samples. The microtippipettor assembly can include a lane elevator, a fluid level sensor, amicrotip mandrel for engaging a microtip 542, a microtip aspirator todrive pipetting action, and a microtip ejector to release microtips fromthe microtip pipettor assembly. In some embodiments, the microtippipettor assembly can access microtips 542 held in a microtip holder onthe cartridge carriage 816, and may return microtips 542 to thecartridge carriage after use. A microtip pipettor assembly may be usedto transfer a reaction vessel plug 222 to a reaction vessel base 246. Insuch an embodiment, the microtip pipettor assembly may also remove aplugged reaction vessel from the assay cartridge, and transport aplugged reaction vessel between different areas of the system.Processing lanes 116 that incorporate a microtip pipettor assembly mayinclude an elution lane 116(e) or other processing lanes where transferof small volumes of liquid is necessary.

In an alternative embodiment, processing lanes 116 may incorporate dualresolution pipette pumps, which are capable of accurate aspiration anddispensing of a wide range of volumes. In some embodiments, pipettingfunctions may be provided by a gantry system that supports one or morepipettor carriages, similar to the pipettor carriage 712 of the samplepipettor 700 that positions a pipettor over a processing lane whenneeded.

FIG. 10( b) shows an example of a processing lane 116 that includes amagnetic separation mechanism that incorporates a separation magnet 804to selectively apply a magnetic field to the contents of a well of theassay cartridge 200, permitting the system to remove liquid contentswithout removing a magnetically responsive solid or particulate phase.Examples of such processing lanes can include an ambient temperaturelane 116(h), a wash lane 116(b), an elution lane 116(e), or otherprocessing lane where manipulation of a magnetically responsive solid orparticulate phase is needed. The applied magnetic field draws themagnetically responsive solid or particulate phase to an internalsurface of the assay cartridge 200 near the region where the magneticfield 804 is applied. In some embodiments, this region is within theculvert 211 at the lower proximal aspect of the reaction well 202. Thispermits a pipettor to enter the reaction well 202 and withdraw liquidcontents at a point distant from the culvert 211, at the point ofgreatest reaction well depth. This relative positioning of a pipettorand the separation magnet 804 advantageously permits the removal of alarge a fraction of the fluid held in the reaction well with minimalrisk of unintended aspiration of the magnetically responsive solid orparticulate phase. Removing a large fraction of fluid is beneficialbecause residual fluid degrades wash efficacy. Retention of asignificant portion of residual fluid within a well may require the useof additional processing steps in order to sufficiently reducecontamination. This in turn requires additional processing time and theconsumption of additional reagents. Separation magnets 804 of differentprocessing lanes 116 may be of different shapes and sizes,advantageously permitting the system to generate “pellets” ofmagnetically responsive solid or particulate phase materials withdifferent sizes and geometries when the field of the separation magnetis applied to an assay cartridge 200, advantageously allowingoptimization of pellet dimensions for specific processing steps. Aseparation magnet may include a backing device that helps shape andfocus the magnetic field. Such backing devices can be made with magneticstainless steel.

Some embodiments of the invention can be directed to a system comprisinga slidable cartridge carriage configured to engage an assay cartridge,the cartridge carriage engaging a carriage track. It can also include aslidable magnet trolley, the slidable magnet trolley engaging thecarriage track and comprising a separation magnet, and a reversiblecoupling device (e.g., a magnet) configured to reversibly coupleslidable cartridge carriage and the slidable magnet trolley. In analternative embodiment, a magnet may be brought into proximity to anassay cartridge using a pivoting mechanism that rotates the magnet intoposition. In another embodiment a magnet may be moved vertically to bebrought into proximity to an assay cartridge. In such an embodiment, themagnet may be coupled to a vertically mounted linear actuator, a railsystem, or other suitable vertical transport.

Illustratively, in some embodiments, each processing lane 116 thatincorporates a separation magnet 804 includes a movable magnet trolley808 disposed to travel parallel to or, along the carriage track 818. Anembodiment of a magnet trolley is shown in FIG. 10( b). By disposing themagnet trolley 808 at different distances from the assay cartridge 200,the system may selectively apply a magnetic field to the contents of theassay cartridge. The magnet trolley 808 may be placed at an end of theprocessing lane that gives it access to the reaction well 202 of anassay cartridge 200 held within that processing lane. Alternatively, thesystem may selectively apply magnetic fields using a controllableelectromagnet proximate the reaction well. In another embodiment, thesystem may selectively apply magnetic fields by moving a magnetic shieldbetween a magnetic field source and the assay cartridge.

In one embodiment, the magnet trolley 808 uses movement of the samecarriage drive used to move the cartridge carriage 816 to apply amagnetic field to the assay cartridge 200. Alternatively, the system maymove the magnet trolley independently of the carriage drive. In someembodiments, the magnet trolley 808 includes a secondary latching magnet812 that couples the magnet trolley 808 to the cartridge carriage 816.The latching magnet 812 is an example of a reversible coupling. Othersuitable reversible couplings may include mechanical devices such aslatches that can be mechanically actuated.

In operation, the system moves the cartridge carriage 816 to a firstposition adjacent the magnet trolley 808, activates a latchingmechanism, and then withdraws the cartridge carriage to the nextoperative location with the magnet trolley in tow. In order to disengagethe magnet trolley 808, the cartridge carriage 816 may be moved to asecond position that aligns the magnet trolley with a locking mechanismthat, when activated, prevents the magnet trolley from moving.Subsequently, moving the cartridge carriage 816 releases the latchingmagnet 812 and removes the assay cartridge 200 from the field of theseparation magnet 804. The first position and the second position may besubstantially identical in some embodiments.

In some embodiments, the latching mechanism includes a latching magnet812 and a magnetically responsive strike plate 814. One of the latchingmagnet 812 and the strike plate 814 may disposed on the magnet trolley808 and the other on the cartridge carriage 816. In some embodiments,the latching magnet 812 is disposed on the magnet trolley 808 to reducethe influence of the magnetic field from the latching magnet on theassay cartridge 200 contents. Alternatively, the cartridge carriage or aportion thereof may be composed of a magnetically responsive material.In some embodiments, the locking mechanism may include a lockingactuator 806, positioned on the lane support 834 so that it can bealigned with the magnet trolley 808. Such a locking actuator 806 may beactivated to fix the magnet trolley 808 to the lane support 834 ordeactivated to permit the magnet trolley to move with the cartridgecarriage 816.

In one embodiment of the operation of the magnetic separation mechanism,the magnet trolley 808 may normally reside in a home position near oneterminus of the carriage track 818. The cartridge pusher may positionthe cartridge carriage 816 adjacent the magnet trolley 808, allowing thelatching magnet 812 to engage the strike plate 814 and thereby attachingthe magnet trolley 808 to the cartridge carriage 816. When attached tothe cartridge carriage 816, the magnet trolley 808 may align theseparation magnet 804 immediately adjacent the reaction well 202,thereby applying a magnetic field to the reaction well contents. Theseparation may be held at an angle that is complementary to that of awall of the reaction well. Subsequent motion by the cartridge pushermoves the cartridge carriage 816 and the attached magnet trolley 808 asa substantially single unit, maintaining proximity of the separationmagnet 804 to the assay cartridge 200 during subsequent processingsteps. Such processing steps may include the removal of liquid from awell of the assay cartridge 200 or dispensing of fluid into a well ofthe assay cartridge.

To detach the magnet trolley 808, the cartridge pusher positions thecartridge carriage 816 such that the magnet trolley returns to its homeposition. The locking actuator 806 may then be activated to engage afeature that prevents the magnet trolley 808 from moving. The cartridgepusher then moves the cartridge carriage 816 away from the homeposition. By arranging the locking actuator 806 to exert a greater forceon the magnet trolley 808 than that of the latching magnet 812 on thestrike plate 814, the motion causes the cartridge carriage 816 toseparate from the magnet trolley. In some embodiments, the lockingactuator 806 is a linear actuator such as a pneumatic cylinder orsolenoid disposed on the lane support 834. The feature that engages thelocking actuator 806 can be a hole or piercing in the magnet trolley 808disposed to align with the locking actuator when the magnet trolley isin its home position.

A consequence of this arrangement of the magnet trolley 808 andcartridge carriage 816 is that the separation magnet 804 can onlyapproach the assay cartridge 200 at the reaction well 202. Thisadvantageously prevents unwanted interactions between the separationmagnet and other assay cartridge compartments, in particular reagentwells utilized for storage of magnetically responsive solid phase ormicroparticles.

As noted above, different processing lanes 116 may utilize separationmagnets 804 with different dimensions. Magnets may be found intemperature stablization lanes, wash lanes, elution lanes, PCR preplanes, transfer lanes, etc. For example, ambient temperature lanes116(h), wash lanes 116(a), and elution lanes 116(e) may use a relativelylarge separation magnet 804. A large separation magnet 804 may apply astronger magnetic field to more rapidly collect magnetically responsivesolid phase or microparticles dispersed throughout a liquid volume, thusreducing time required for processing. A large separation magnet 804 mayapply a magnetic field to collect magnetically responsive solid phase ormicroparticles from the reaction well contents onto a relatively a largearea of the reaction well 202 inner surface. This large areaadvantageously disperses the magnetically responsive solid phase ormicroparticles, reducing the opportunities for interaction between themso that subsequent resuspension of the magnetically responsive solidphase or microparticles may be less vigorous and more complete. This inturn reduces the time required for processing and reduces the chances ofcontamination resulting from fluids that might remain trapped withinclumps of aggregated material.

Other processing lanes 116, such as certain wash lanes 116(b), may use arelatively small separation magnet 804. A small separation magnet 804concentrates the magnetic field on a relatively small area of surface ofthe assay cartridge. In some embodiments, the small area may overlap thearea of the reaction well 202 that is affected by a large separationmagnet 804 and is disposed close to the bottom of the reaction well. Asmall separation magnet 804 advantageously supports processing stepswhere it is desirable to collect magnetically responsive microparticlesin a small area. Such processing steps include resuspension of themagnetically responsive solid phase or microparticles in a relativelysmall volume of fluid. For example, elution of nucleic acids from themagnetically responsive solid phase or microparticles using a very smallvolume of fluid allows the system to effectively concentrate theresulting eluted nucleic acid as described below. Processing in a washlane 116(b) may precede elution in many protocols so that the relativelysmall eluent volume may more readily re-suspend the collectedmicroparticles.

Processing lanes 116 may also include features used to confirm thealignment of various lane components. Such features may includealignment flags. In FIG. 10( c), a first alignment flag 900 attached tothe cartridge guide 800 and a second alignment flag 897 attached to theattached to the cartridge carriage 816 are shown. These alignment flagsare described in further detail below.

Consistent processing of samples can necessitate control of thetemperature of assay cartridge 200 contents during processing. Toaccomplish this, processing lane 116 may include a heating assembly, forexample, a lane heater, of varying configuration. With reference toFIGS. 10( d)-11, some processing lanes 116 may include a lane heater840, 1103 that heats at least a portion of the assay cartridge 200. Thelane heater 840, 1103 may heat the reaction well 202, as shown in FIGS.4( a) and 10(b), wells used for storing assay reagents 204, 208, 209, ora combination of these, as shown in FIG. 10( d). This advantageouslypermits the performance of specific processing steps at elevatedtemperatures, if desired, and may allow pre-heating of reagents prior toaddition to the reaction well 202 in order to tightly control reactiontemperature. In some embodiments, the reaction well 202 and the largereagent wells 204 are heated. The lane heater 840, 1103 may be disposedat the proximal end of the lane motion path and configured so that thecartridge carriage 816 can drive the assay cartridge 200 into the laneheater 840, 1103. In one embodiment, the lane heater 840 or a portionthereof may be of floating clamshell construction, with two independentsides configured to fit snugly around an end of the assay cartridge 200,and an open end to permit entry of the assay cartridge. The lane heater840, 1103 may have an open top 850 to accommodate the reaction well 202.In some embodiments, the two independent sides each contain a heat block854 to provide heat, at least one temperature sensor 860 to control theheater temperature, an insulated cover 856 on the external aspect tocontain heat, and a spring to couple the independent sides against theassay cartridge 200. The two heat blocks 854 may couple to one anotherin a pivoting connection 858 at the end opposite the open end 852. Thecavity between the heat blocks 854 may be slightly narrower than thewidth of the reaction well 202 so that the spring drives the two heatblocks 854 into tighter thermal contact with the assay cartridge 200walls.

In an embodiment shown in FIG. 11, the lane heater 1103 has two heatingdevices 1104 and 1106, with one heating device 1104 that applies heat tothe reaction well 202 and a second heating device 1106 that applies heatto reagent storage wells 204 of an inserted assay cartridge 200. Theheating devices 1104 and 1106 may be configured so that the heatingsurfaces do not contact the assay cartridge but are in close proximity,providing heat via radiation and convection. Alternatively, the reactionwell heating device 1104 may be configured similarly to the lane heater840 shown in FIG. 10( c), which contacts the exterior wall of thereaction well 202 and is described in detail below. These heatingdevices may act in concert or be controlled independently.

The lane heater 840 may mount to the lane support 834 by a floatingconnection so that slight misalignment or flexure of the assay cartridge200 does not impede insertion into the lane heater. The tapered shape ofthe reaction well 202, which may be mirrored by an internal contour ofthe lane heater 840, further serves to guide insertion. The cartridgeguide 800 terminates distal to the lane heater 840 so as not tointerfere with insertion.

In some embodiments, in operation, the cartridge pusher moves thecartridge carriage 816 towards the lane heater 840 so that the leadingedge of the reaction well 202 engages the corresponding taper in theheat block 854. As the reaction well enters further, the side walls ofthe reaction well 202 engage the internal walls of the heat block 854,enlarging the cavity by pivoting the heat blocks about their connectionpoint 858. The heat block 854 position adjusts to press inward on theexternal walls of the reaction 202 when the assay cartridge 200 iscompletely inserted. The lane heater 840, 1102 may maintain temperatureby any of a number of methods, but the temperature may be maintained bycontrolling the heaters with a PID loop connected to the temperaturesensors 860. The cartridge pusher may disengage the assay cartridge 200from the lane heater 840 by simply repositioning the cartridge carriage816 in the distal direction.

The efficiency of an instrument process may be affected by thetemperature of the testing environment. The testing environment mayimpact both the temperature of the contents of the assay cartridge 200(held in storage prior to use) and the temperature of the samples beingprocessed. For example, the efficiency or reproducibility of chemistryprocesses may be negatively impacted if the samples that are beingprocessed are too cold. Heaters may be integrated into lane designs thatrequire access to assay cartridge 200 contents (as discussed above), butwhile such heaters may be adequate to maintain the temperature of anassay cartridge, they may not be sufficient to bring assay cartridgecontents from ambient to processing temperature within a single pitchinterval. Thus, in some embodiments of the invention, an instrument orprocess disclosed herein further includes one or more direct anddedicated heating components or steps for this purpose. For example, aninstrument may include one or both of a cartridge heater coupled to anassay cartridge to raise the temperature of an assay cartridge and itscontents and one or more lane heaters integrated into processing lanesto maintain the temperature of an assay cartridge and its contents.

An instrument disclosed herein may include one or more cartridgeheaters, configured to transfer heat to an assay cartridge 200, therebytransferring heat to a sample and other liquid components contained inan assay cartridge. The cartridge heater may be under active control,such that heat applied to an assay cartridge is controlled by acontroller running computer software. For example, the controller mayaccess a protocol specifying, for one, some or all assay cartridges: adesired sample or reagent temperature or temperature range, a desiredsample or reagent temperature profile (e.g., that the sample be warmedfrom a first temperature to a second temperature over a given period oftime or during a certain processing stage), or an output of a cartridgeheater, advantageously allowing the system to perform a broad range oftemperature dependent processes. For example, a protocol may requirethat a first step be performed at an elevated temperature, for examplethe lysis of gram positive bacteria, that is incompatible with processesperformed at other steps. Such protocols may perform the first step in afirst processing lane and the second step in a second processing lane.In one embodiment of such a protocol a first step may be performed at60° C. to 80° C. and a second step at 30° C. to 50° C. In anotherembodiment of such a protocol a first step may be performed at 65° C. to75° C. and a second step at 35° C. to 45° C. In still another embodimentof such a protocol a first step may be performed at about 70° C. and asecond step at about 37° C. If a protocol requires a certaintemperature, the controller, using the computer software, may determinea voltage or a voltage temporal profile to be provided to one or morecartridge heaters. Such a determination may be based, e.g., uponmeasured temperatures of an assay cartridge or sample or reagentstherein, physical characteristics of an assay cartridge (e.g., a size,shape or material), a specific heat of a reagent or sample, a startingtemperature of a reagent or sample, and/or an ambient temperature.

FIG. 20( a) shows an embodiment of a cartridge heater 3005. Thecartridge heater may be an example of a heating assembly. The cartridgeheater 3005 may be configured to apply heat to one or more sides of anassay cartridge 200. The cartridge heater 3005 may comprise a front wall3007(a) and a back wall 3007(b), as shown in FIG. 20( b). The front wall3007(a) may be positioned adjacent to a first side of the assaycartridge 200, and the back wall 3007(b) may be positioned adjacent to asecond side of the assay cartridge 200 opposite the first side. Thefirst and second walls 3007(a) and 3007(b) may be connected, e.g., by atop wall 3007(c). As shown in FIG. 20( a), the top wall may include ahinge that permits the front wall 3007(a) to pivot relative to theheater back wall 3007(b). The cartridge heater 3005 may also includemount elements 3010 in FIG. 20( a), which include spring mounts that canbe seen protruding through the wall 3007(a) of the heater in FIG. 20(b). These serve to press the right hand interior heater component 3027against the outer wall of the assay cartridge 200, and therefore topress the assay cartridge 200 against the left hand interior heatercomponent.

The cartridge heater 3005 may be moved between open and closed positionsby heater actuator 3015, as shown in FIGS. 20( c) and 20(d). The heateractuator 3015 may be a linear actuator. The cartridge heater's back wall3007(b) may be substantially fixed in position. The instrument maydetermine that a cartridge 200 has been moved into a heating positionbetween the front and back walls 3007(a) and 3007(b). For example, thecontroller may sense the assay cartridge 200 (e.g., via an opticaldetector or a movement detector) or it may receive a signal indicatingthe cartridge's new presence. The controller may determine whether thecartridge heater 3005 is in an open position or a closed position (e.g.,using a sensor). The front wall 3007(a) is further from the cartridge200 and the back wall 3007(b) in the open position as compared to theclosed position. If the cartridge heater 3005 is in an open position,the heater actuator 3015 may move a portion of the cartridge heater 3005(e.g., the front wall 3007(a)) to a closed position closer to the assaycartridge heater. In some instances, the front surface 3007(a) is incontact with the assay cartridge in a closed position but not in an openposition.

FIGS. 20( c) and 20(d) show an embodiment in which the actuator 3015moves the front wall 3007(a) angularly to reduce an angle between thefront and back walls 3007(a) and 3007(b). Thus, the front wall 3007(a)moves closer towards the lane's center and clamps onto the cartridge200. The heater 3005 may then be in close thermal contact with the assaycartridge 200 and heat the assay cartridge 200 using both the front andback walls 3007(a) and 3007(b). Since the walls 3007(a), 3007(b) can bein physical contact with the assay cartridge 200, heat can be quicklytransferred to liquids in the cartridge 200 by thermal conduction. Insome embodiments, the actuator 3015 moves the front wall 3007(a)horizontally and/or vertically.

FIG. 20( e) shows a section of an embodiment of a cartridge heater 3005.As shown, the cartridge heater 3005 may include a plurality of heaterzones. The heater zones may correspond to different portions of an assaycartridge 200. For example, the cartridge heater 3005 may include afirst heater zone 3005(a) configured to heat large reagent wells 204 anda second heater zone 3005(b) configured to heat medium reagent wells 209of the assay cartridge 200. By including different zones, samples andreagents deposited into different wells of a cartridge can be raised todifferent temperatures. Additionally, the zones may permit the wells tobe raised to the same temperature (e.g., by accounting for well shapesand/or relative locations of wells within the cartridge). A zone may beconfigured to provide substantially uniform heat throughout the zone, toprovide varying heat across the zone (e.g., to apply more heat to outerzone portions than middle portions), or to provide heat in discreteregions.

The cartridge heater 3005 may comprise a plurality of heating elements3020. Each heating element 3020 may be sized and position to heat one ormore wells in the cartridge 3200. Each heating element 3020 may be underseparate control, such that it can produce independent heating output.

FIG. 20( f) shows components of a cartridge heater 3005. As describedabove, the cartridge heater 200 may include a front wall 3007 a and aback wall 3007 b. Each wall may include a heater casing 3025. The heatercasing 3025 may partly encapsulate an interior heater component 3027.The interior heater component 3027 may be connected to a heater casing3025 using one or more connectors 3010, as shown in FIG. 20( e). Theinterior heater component 3027 may include one or more heating elements3020. The casing 3025 may prevent heat from the heating elements 3020from escaping in a direction not in the direction of the cartridge 200.It may also reflect heat to improve the efficiency of the cartridgeheater 3005.

The heating elements 3020 may be partly covered by an insulator 3017,such as a foam insulator. The insulator 3017 may comprise holes, inwhich may reside a thermal cut off element 3012 (see below). The holescan provide access for other system components or to allow heat producedby the heating elements 3020 to be disbursed primarily in discrete andtargeted locations. One or both of the interior heater components mayinclude one or more thermistors (not shown). The thermistors may monitorthe temperature of the interior heater component 3027, and an output ofthe heating element 3020 may be adjusted based on the monitoredtemperature. The thermal cut off element 3012 can be a temperaturesensitive switch that acts as a local safety feature by stopping powerto the heating element should the temperature exceed a pre-set limit.

FIG. 20( g) shows a portion of an embodiment of an assay cartridge 200that may be used with the cartridge heater 3005. The assay cartridge 200includes large reagent wells 204 and medium reagent wells 208 but nosmall reagent wells. The assay cartridge also includes reaction vesselcomponent holders 219. The wells 204 and 208 may have a cross-sectionwith a substantially flat and vertical side along the long side of theassay cartridge 200. For example, the wells 3204 and 3208 may have asubstantially rectangular cross-section. This may increase the surfacearea facing the cartridge heater 3005 and thereby increase heatingefficiency. The interior heater components 3027 may be configured tocontact a flat external surface of the large and medium reagent wells204 and 208. In some instances, the reaction vessel component holders219 do not include a side that is substantially flat and vertical. Thus,there may be nominal clearance between the reaction vessel componentholders 219 and the cartridge heater 3005 during heating.

All wells corresponding to a particular heating zone may have asubstantially similar size, shape and/or heater-adjacent surfaceprofiles. This may allow the wells to be evenly heated by a uniform heatoutput by a heating zone. For example, an assay cartridge 200 mayinclude a plurality of large reagent wells 204, and a cartridge heater3005 may include a first heating zone 3005 a with an area and positioncomplementary to a side-surface area of a large-well portion of thecartridge 200. The first heating zone can be juxtaposed with thereaction well in the assay cartridge in some embodiments. Similarly, anassay cartridge 200 may include a plurality of medium reagent wells 208,and a cartridge heater 3005 may include a second heating zone 3005 bwith an area and position complementary to a side-surface area of amedium-well portion of the cartridge 200. The second heating zone can bejuxtaposed with a reagent well in the assay cartridge.

The cartridge heater 3005 in FIGS. 20( a) and 20(b) is in a relativelyfixed position within an instrument, only moving relatively smalldistances towards and away from the center of a lane. In someembodiments, a cartridge heater 3005 moves along with an assay cartridge200 as the assay cartridge 200 progresses through different lanes andprocessing stages. For example, a cartridge heater 3005 may bepositioned on a top surface of the assay cartridge 200 after samplesand/or reagents have been added to the wells.

FIG. 20( h) shows a top plan view of a layout of the components of aninstrument according to an embodiment of the invention, with somecomponents removed clarify the basic structural and functional modules.Many of the instrument's lanes, units and components parallel those inabove-described embodiments and like numerals can refer to likefeatures. Thus, above-described details of similar components may alsopertain to the lanes, units and components depicted in FIG. 20( h).

The layout shown in FIG. 20( h) includes a cartridge warming lane3116(i). In this lane, one or more assay cartridges 200 may be warmed byone or more cartridge heaters 3005, as described above. The heating lane3116(i) may include a pump to transfer fluids (e.g., samples) from onewell to another.

In some embodiments, one or more lane heaters 3040 (distinct from thecartridge heater 3005) are integrated into one or more processing lanesand cartridge loading lanes. Lane heaters 3040 may be configured toprimarily maintain a temperature of an assay cartridge and/or itscontents and/or to regulate the temperature within a small rangerelative to the cartridge heater's range of regulation. Thus, acartridge heater 3005, which may contact or be very close to a largesurface area of the assay cartridge 200, may quickly and reliablyinitially heat the assay cartridge 200. Lane heaters 3040, which may bepositioned further from the assay cartridge 200, may then be tasked withtemperature regulation within a smaller range of temperatures. In someinstances, a cartridge heater 3005 is configured to heat an assaycartridge 200 primarily by conduction, while a lane heater 3040 isconfigured to heat an assay cartridge 200 primarily by convection and/orradiation. Thus, the cartridge heater 3005 may heat the assay cartridge200 faster, more efficiently and more reliably than a lane heater 3040can. Despite the structural and efficiency advantages of using acartridge heater 3005, in other embodiments, an instrument includes onlylane heaters 3040 and no cartridge heater 3050.

Lane heaters 3040 may be included in one, more or all of the lanes(e.g., shown in FIG. 1( b) or FIG. 20( g)). In some embodiments, elutionlane 116(e), wash lanes 50, 116(a) and 116(a)′, and temperaturestabilization lane 116(j) include a lane heater 3040. Lane heaters 3040may be structurally the same or similar across lanes. In some instances,lane heaters 3040 differ across lanes, e.g., based on prior, current orsubsequent processing. For example, the size, number of position of alane heater's heating elements 3020 may vary depending on which wellsare likely to have contents in the lane. Such heating-elementspecificity may reduce system noise and improve system power efficiency.

FIGS. 20( j) and 20(k) show embodiments of an instrument with a laneheater 3040. The lane heater 3040 may comprise structural parts and/orcharacteristics similar to or the same as those described with respectto the cartridge heater 3005. As shown in FIG. 20( j), the lane heater3040 may be positioned substantially under the cartridge guide 800, suchthat the interior heater components 3027 may heat the wells of the assaycartridge 200. In some embodiments, interior heater component 3027 isfixed and positioned to straddle the sides of the cartridge 200.Therefore, unlike the cartridge heater 3005, the lane heater 3040—insome instances—may not include an actuator 3015 to move one of the laneheater's walls. Rather than clamping onto an assay cartridge 200, thelane heater 3040 may be positioned and configured to be near the sidesof the assay cartridge 200. In some embodiments, the lane heater 3040 isnot in direct contact with the assay cartridge 200 (i.e., a gap existsbetween the interior heater components 3027 and the cartridge 200).

Though the heat transfer to the assay cartridge 200 may be lessefficient, this configuration eliminates the need to have a movingheater part, thereby reducing potential mechanical difficulties, spacerequirements and processing time. Thus, an assay cartridge 200 may movealong the cartridge guide 800 down the lane until it is positionedbetween walls of the lane heater 3040. The lane heater 3040 may adjustor maintain the assay cartridge's temperature to or within a desiredrange while or before the appropriate processing is occurring.

In some embodiments, the above-described cartridge heater 3005 and/orlane heater 3040 may be configured to cool a cartridge and/or itscontents. For example, the heating elements 3005 may be replaced withcooling elements that may cool a nearby or in-contact cartridge 200using cycled chilled fluid and/or thermoelectric cooling

While the above describes several heater designs based on resistanceheaters, other embodiments may incorporate alternative heating methodsto accomplish the same ends. Such heating methods include infraredheaters, convection or forced air heaters, Peltier devices, and flexibleheaters that conform to the surface of the assay cartridge 200.Alternatively, liquids may be heated within pipette tips prior to beingdispensed.

Processing lanes 116 may provide access for processing tools on thesystem that are external to the processing lanes so that they mayoperate on assay cartridges 200. For example, as shown in FIG. 1( b) thecartridge loading lane 116(f) may receive assay cartridges 200 from thecartridge loading unit 112 and may present the received assay cartridgeto the sample pipettor 70 for addition of sample, and to the XYZpipettor on the XYZ transport device 40 for addition of reagents fromreagent packs 400. The elution lane 116(e) may exchange microtips 542with the XYZ pipettor on the XYZ transport device 40. The amplificationpreparation lane 116(g) may present the assay cartridge 200 to the XYZpipettor on the XYZ transport device 40 for transfer of materialsbetween compartments, for addition of reagents from reagent packs 400,for plugging of reaction vessels 221, and for removal of reactionvessels. The waste lane 116(c) may transfer liquid contents of the assaycartridge 200 to liquid waste storage 94 and may move the expended assaycartridge to solid waste storage 92 as shown in FIG. 1( d).

Processing lanes 116 may perform any available operation on an assaycartridge 200 present in the processing lane during a fixed or specifiedoperational interval, or “pitch”. An operation is available if theprocessing lane 116 has access to processing tools needed for theoperation. Some operations, such as simply storing an assay cartridge200 during an extended reaction, require no processing tools. Others,such as transfer of materials between compartments of an assay cartridge200, may need access to processing tools that may be resident in theprocessing lane 116. Still other operations, such as transfer ofreagents from outside of the assay cartridge 200, may require access toprocessing tools external to the processing lane 116. Since suchexternal processing tools may be otherwise engaged such operations mayintroduce constraints on the flexibility of processing lane operationscheduling; a processing lane 116 has access to an external processingtool only while that tool is not being utilized for other tasks. In someembodiments, different types of processing lanes 116 may have access toprocessing tools as described below.

The cartridge loading lane 116(f) may have access to the cartridgeloading unit 112, to the sample pipettor 70, to the XYZ pipettor on theXYZ transport device 40, and to the transfer shuttle 50. Availablefunctions of the cartridge loading lane 116(f) can include loading assaycartridges 200 from the cartridge loading unit 112 and presenting thosecartridges for resuspension of solid phase, microparticle or lyophilizedreagents, fluid addition, piercing of the barrier film 205, and mixingby the sample pipettor 70 and the XYZ pipettor on the XYZ transportdevice 40. Either the sample pipettor 70 or the XYZ pipetter on the XYZtransport device 40 may transfer a fluid to, from, or within an assaycartridge 200 in the cartridge loading lane 116(f). The cartridgeloading lane 116(f) may share an extended cartridge pusher with thecartridge loading unit 112. At the intersection of the sample pipettor70 motion path, the cartridge guide 800 in the cartridge loading lane116(f) may have an opening or gap to admit the sample pipettor 70. At aposition accessible to the XYZ pipettor on the XYZ transport device 40,the cartridge guide 800 in the cartridge loading lane 116(f) may have anopening or gap to admit the XYZ pipettor.

A high temperature stabilization lane 116(j) may have access to a laneheater (840, 1103), to a millitip pipettor 704, and to the transfershuttle 50. Available functions of a temperature stabilization laneinclude heating assay cartridge 200 contents, microparticle or solidphase resuspension, mixing, and transfer of materials among compartmentsof an assay cartridge.

A low temperature stabilization lane 116(h), which may provide heat at alower temperature than the high temperature stabilization lane 116(j),may have access to a millitip pipettor 704, to a separation magnet 804,and to the transfer shuttle 50. Available functions of a low temperaturestabilization lane (e.g., an ambient temperature lane) 116(h) includere-suspension of microparticles or solid phase reagents, mixing, andtransfer of materials among compartments of an assay cartridge 200.Additionally, the low temperature stabilization lane (e.g., an ambienttemperature lane) 116(h) may apply a magnetic field to the assaycartridge 200 to facilitate separation and washing of magneticallyresponsive solid phases or microparticles.

A wash lane 116(b) may have access to a millitip pipettor 704, to aseparation magnet 804, and to the transfer shuttle 50. The separationmagnet 804 of a wash lane 116(b) may be smaller than the separationmagnet of a low temperature stabilization lane 116(h). Availablefunctions of the wash lane 116(b) include re-suspension ofmicroparticles or solid phase reagents, mixing, and transfer ofmaterials among compartments of an assay cartridge 200. Additionally,the wash lane may apply a magnetic field to the reaction well tofacilitate separation and washing of magnetic microparticles. Wash lanesin general may include large or small magnets.

An elution lane 116(e) may have access to a microtip pipettor 1142similar to that utilized by the XYZ transport device 1100 of FIGS. 15(a)-15(c), to a separation magnet 804, to an XYZ pipettor on an XYZtransport device 40, and to a transfer shuttle 50. It can also disposeof microtips within used wells of the assay cartridge. Availablefunctions of the elution lane include re-suspension of microparticles,mixing, and transfer of materials among compartments of an assaycartridge. Additionally, an elution lane 116(e) may apply a magneticfield to the assay cartridge 200 to facilitate collection of suspendedmagnetically responsive solid phases or microparticles. The elution lane116(e) can also have the capability to pick up, drop off, and seat thevessel plug 222 to close the reaction vessel 221. Because it providesaccess to the XYZ pipettor 40, the elution lane 116(e) may transfermaterials between the assay cartridge 200 and the reagent storage unit124 and transfer materials between the assay cartridge and any of thethermal cycler modules 1300 (see FIG. 16( a)). The elution lane 116(e)can have a source of and disposal method for microtips 542. In someembodiments, microtips are disposed of by ejection into a well of anassay cartridge 200. In other embodiments, the XYZ pipettor on the XYZtransport device 40, which has access to both source and disposal sitefor microtips 542, delivers one or more microtips 542 to the elutionlane. After the microtip pipettor in the elution lane uses the microtips542, the XYZ pipettor on the XYZ transport device 40 may pick up andthen discard the expended microtips 542.

An amplification preparation lane 116(g) may have access to the XYZpipettor of the XYZ transport device 40 and the transfer shuttle 50.Available functions of the amplification preparation lane 116(g) mayinclude re-suspension of microparticles or solid phases, mixing, andtransfer of materials among compartments of an assay cartridge 200,transfer of materials between the assay cartridge and the reagentstorage unit 124, and transfer of materials between the assay cartridgeand any of the thermal cycler modules 1300. Additionally, the XYZpipettor of the XYZ transport device 40 may pick up, drop off, and seatthe vessel plug 222 to close the reaction vessel 221 and to transportthe reaction vessel. The cartridge guide 800 of the amplificationpreparation lane may have an opening or gap at a location within thereach of the XYZ pipettor of the XYZ transport device 40 in order toadmit the XYZ pipettor. The amplification preparation lane 116(g) mayhave a vessel detection sensor, which can sense the conductive plug of asealed reaction vessel. Such a vessel detection sensor may utilize aliquid level sensing circuit to detect the presence of a conductiveplug. Alternatively, the vessel detection sensor may utilize a pressuresensor that monitors the internal pressure of the pipette pump. Asanother alternative, the vessel detection sensor may utilize both aliquid level sensing circuit and a pressure sensor to detect thepresence of a sealed reaction vessel on a pipette mandrel. Theamplification preparation lane 116(g) can also have a connection to thewaste chute-utilized to collect microtips and used (i.e. after thermalcycling) reaction vessels. The XYZ gantry can utilize a “soft eject”routine that slowly eases these items off of the pipette mandrel so thatthey drop in a controlled manner.

The waste lane 116(c) can includes access to an aspiration probe 986, toa solid waste ejector 874, and to a transfer shuttle 50 as shown inFIGS. 14( a) and 14(c). Available functions of the waste lane 116(c)include draining liquids from assay cartridge 200 compartments anddisposal of assay cartridges.

Subject to the conflict constraints on the use of external tools, and tothe timing constraints of the pitch interval and transfer windows asdiscussed below, a processing lane may perform any available operationin any sequence. A first protocol and a second protocol may specify thatthe same operations are performed in a given processing lane 116, or thefirst protocol may specify operations in a given lane that differ fromthose specified by a second protocol. This processing lane conceptprovides capabilities for flexible protocol execution by a combinationof this selectable operation sequence within a processing lane and bythe ability to route an assay cartridge through selectable sequence ofprocessing lanes.

Other embodiments of the invention can include a number of otherfeatures, in addition to or as alternatives to the features describedabove. For example, embodiments of the invention may one or moremultifunctional lanes, each lane capable of performing all sampleprocessing steps on an inserted cartridge. Such a processing lane mayinclude a thermal cycler module.

K. Microtips

FIG. 12( a) shows a side, cross-sectional view of a pipettor mandrel 460engaged with a collar 490(a) of a microtip 490. FIG. 12( b) shows aperspective view of the microtip 490 shown in FIG. 12( a).

In embodiments of the invention, a microtip 490 can be a relativelysmall-capacity pipette tip, e.g., having a capacity no greater thanabout 100 or 2004. The microtip 490 may be used for one or more the usesdescribed above with respect to millitip 220, such as for use during theisolation phase.

The microtip 490 may share any or all of the physical characteristicsdescribed above with respect to millitip 220. For example, the microtip490 may taper to a pipetting orifice and may couple to a pipettorthrough a compliant coupling taper supporting remove-and-replaceoperations. A length of the microtip 490 may be sufficient to reach thedepth of a 100 mm tube or other sample containers used on the systemwhen mounted on a suitable pipette mandrel. In some embodiments, thelength of the microtip 490 is about 30-80 mm, e.g., about 50 mm.

As shown in FIGS. 12( a) and 12(b), the microtip 490 can include amounting aperture that couples to a pipettor mandrel 460 during use. Themicrotip 490 may be tapered, e.g., in a plurality of segments. Thus, acoupling taper 490(a) may extend from a mounting aperture to a lowerdiametral step forming a seating surface 490(a)-2. As with the millitip220, the microtip 490 may include an upper taper 490(e), a middle taper490(d), and a lower taper 490(c). These taper segments may have one ormore described above with respect to the millitips' respective segments.In some embodiments, for the microtip 490, the middle taper 490(d) (notthe upper taper 490(e)) extends for the majority of the part length, asshown in FIG. 12( b). In some embodiments, the coupling taper 490(a)extends about 5-15 mm (e.g., about 7.5 mm) from the top of the microtip,the upper taper 490(e) extends about 5-15 mm (e.g., about 7.2 mm) fromthe end of the coupling taper, the middle taper 490(d) extends about15-45 mm (e.g., about 28.8 mm) from the end of the upper taper, and thelower taper 490(c) extends about 3-10 mm (e.g., about 6.3 mm) from theend of the middle taper.

The lower taper 490(c) may form the apical end of part that interminates in an annulus (e.g., a flat annulus with a diameter of about0.5 mm to about 1 mm) surrounding a pipetting orifice (e.g., with adiameter of about 0.1 mm to about 0.5 mm). In some embodiments, thediameter of the annulus is about 0.8 mm and the diameter of the orificeis about 0.3 mm.

As with the millitip 220, the coupling taper 490(a) of the microtip 490may be a compliant taper with a smooth interior surface and withoutsupporting ribs. Walls of the coupling taper 490(a) may have a thicknessof about 0.1-1.0 mm (e.g., about 0.5 mm).

As with the millitip 220, the microtip's coupling taper 490(a) mayabruptly change diameter at the top of the upper taper forming a seatingsurface perpendicular to the axis of the microtip 490. The seatingsurface may form a flat annulus having a width of about 0.05-0.5 mm(e.g., about 0.10 mm) surrounding a core having a having a diameter ofabout 1 mm-5 mm (e.g., about 3 mm).

The open end of the coupling taper 490(a) forming the mounting aperturemay end in a stopping annulus, as described above for the millitip 220.The microtip 490 may include an aerosol barrier and/or an abruptinternal diametral decrease in the upper taper 490(a), as describedabove with respect to the millitip 220.

As shown in FIG. 12( c)-1, the microtip may also include one or moreventing features 491 at a lower taper 490(c). FIG. 12( c)-2 shows a sideview of a portion of the lower taper 490(c). The dimensions shown inFIG. 12( c)-2 are in inches, but the dimensions can vary in otherembodiments. In embodiments of the invention, the venting features maycomprise abrupt deviations from the microtip's otherwise smooth outsidewall. The deviations may extend in the vertical direction along a majoraxis of the microtip, and may include sharp corners on the outsidediameter, protruding ribs, incised channels, or similar features. Inaddition, the exterior of the microtip pipette orifice may be anannulus, the plane of which is at right angles to the central axis ofthe microtip. In some embodiments, one or more ventilation features orchannels do not extend to the distal tip of the microtip. For example, aventilation channel may end between about 0.1-0.5 mm (e.g., about 0.25mm) from the end of the tip.

The microtip 490 may comprise one or more materials (e.g., an admixtureof a base polymer with a conductive material) or properties (beingelectrically conductive) as described above with respect to the millitip220. The microtip 490 may be manufactured using a forming process asdescribed above with respect to the millitip's formation.

L. Microtip Storage

FIG. 13( a) shows a front perspective view of a microtip storage unitaccording to an embodiment of the invention, with an access cover in anopen configuration.

FIG. 13( b) shows a portion of a microtip storage unit according to anembodiment of the invention.

FIG. 13( c) shows a top plan view of a portion of a microtip storageunit.

FIG. 13( d) shows a rack clasp in a microtip storage unit according toan embodiment of the invention.

FIG. 13( e) shows a perspective view of a microtip rack according to anembodiment of the invention.

FIG. 13( f) shows an exploded view of a microtip rack according to anembodiment of the invention.

As shown in FIG. 13( f), microtips 542 may be provided in the form of aplurality of tips held in a microtip rack 550. Microtip racks 550 may,in turn, be stored on the system in a microtip storage unit 120. In someembodiments, the microtip rack 550 and the microtip storage unit 120have structural similarities to the reagent pack 400 and the reagentstorage unit 124 (see FIGS. 9( a)-9(e) and FIGS. 8( a)-8(c),respectively).

Referring to FIG. 13( a), the microtip storage unit 120 may include aplatform accommodating one or more microtip racks 550. Storage ofmultiple microtip racks 550 advantageously permits replacement of spentmicrotip racks 550 without interrupting system operations. In oneembodiment, the microtip storage unit 120 accommodates up to fourmicrotip racks 550. This advantageously allows the system to use all ofthe microtips 542 in a single microtip rack 550 without concern thatinsufficient microtips 542 will remain for assays in progress. Themicrotip storage unit 120 may include a conductive path between systemground and any loaded microtip racks 550. This advantageously dissipatesstatic charges that might otherwise accumulate and displace microtips542 from the microtip racks 550. To support this function, at least aportion of the microtip rack 550 may be made of a conductive orantistatic plastic. Such conductive or antistatic plastics includecarbon-filled polypropylene, polyacetylene, polypurrole, polyaniline,and polymers mixed or treated with antistatic agents such as aliphaticamines, aliphatic amides, quaternary ammonium salts, phosphoric acidesters, polyols, polyol esters, PEDOT:PSS, and polyaniline nanofibers.

Each rack 550 may hold any suitable number of microtips. In someembodiments, each rack may hold a 6×20 array of microtips. The racks mayhold more or less microtips in other embodiments of the invention. Insome embodiments, microtips held in a microtip rack 550 may be nestedwithin one another.

As shown in FIG. 13( a) and FIG. 13( b), in some embodiments, themicrotip storage unit 120 can include three or more (e.g., four or more)parallel slots defined by slot walls 520, a rear wall 558, a fingerguide 532, and an access cover 556. Each of the parallel slotsaccommodates a microtip rack 550. A dampening spring may be added to theaccess cover 556 to control the movement of the access cover 556.

The microtip storage unit may also include a base plate 522perpendicular to and connecting the parallel slot walls 520 and the rearwall 558. Each slot may include rack guides 530 extending from the slotwalls 520 to support the lower aspect of the microtip rack flange 560.Rack guides 530 on either side may thus support each microtip rack 550.One or more bias springs 528 (or other type of biasing element) on oneside of each slot may force the microtip rack 550 against the oppositeslot wall 520 to stabilize the microtip rack 550 and assure positionalaccuracy. The front edge of the rack guides may include lead-in features526 that serve to direct microtip racks 550 to compensate formisalignments during the loading process. An exterior wall of themicrotip storage unit may serve as a mounting point for a waste chuteleading to a waste disposal area.

Referring to FIG. 13( c), the rear wall 558 may include a centering pin534 that engages a centering slot 536 on the microtip rack 550 uponinsertion. This centering pin 534 can serve to fix the location of themicrotip rack 550 within the microtip storage unit 120. The microtipstorage unit 120 may further secure each loaded microtip rack in placeusing a spring-loaded rack clasp 554 (FIG. 13( d)), which is similar tothe RSU latch assembly as shown in FIG. 8( c). In some embodiments, therack clasp 554 pivots on a clasp pivot 570 and may engage acomplementary clasp recess 552 in the microtip rack 550 (FIG. 13( e)).The rear wall 558 may also include an ejection spring (or other biasingelement) so that the microtip rack 550 can be ejected when the clasp 554does not secure the microtip rack 550.

A single latch pivot 570 may extend across the rear wall 558 to mount aplurality of rack latches 554 within the microtip storage unit 120. Therack latch 554 may operate in substantially identical fashion to thereagent storage unit 124, seating within a mating feature, such as latchrecess 552, upon loading the microtip rack 550 and holding the microtiprack 550 in place until released. The microtip rack 550 may be releasedfrom the microtip storage unit 120 by the application of downwardspressure on the rack latch tab 568, which causes the rack latch 554 torotate around the axis defined by the latch pivot 570 therebywithdrawing the rack latch 554 from the clasp recess 552 of the microtiprack 550. In one embodiment, the downwards pressure is supplied by theXYZ elevator 1120 (shown in FIG. 15( c)), as applied through a microtip542. As described for the reagent storage unit 124 above, the rear wallof the microtip storage unit 120 may include apertures that align withthe stored microtip racks 550, which would permit spent microtip racksto be displaced out through the rear of the microtip storage unit uponloading of a new microtip rack into the same slot.

The microtip storage unit 120 may include sensors that detect thepresence of microtip racks 550. Suitable sensors include but are notlimited to Hall effect sensors, optical sensors, or gravimetric sensors,and may be affixed to the rear wall 558 of the microtip storage unit120. In one embodiment, the sensor is an optical sensor, such as theOpto slot sensor, from Optek of Carrollton, Tex. Alternatively, thesystem may detect the presence of a microtip rack 550 by confirmingsuccessful loading of a microtip 542 to an XYZ pipettor (e.g., element1136 shown in FIG. 15( a)).

As shown in FIG. 13( b), a finger guide 532 may extend across the upperaspect of the front portion of the microtip storage unit 120. This canact as a guide and as a physical limit during the loading process. Inone embodiment, in order to load a microtip rack 550 into the microtipstorage unit 120, the user slides a microtip rack 550 into a parallelslot by aligning the distal end 566 of the microtip rack 550 above therack guide 530 but below the finger guide 532. As shown in FIG. 13( a),an access cover 556 may shield the front portion of the microtip storageunit 120. In one embodiment, the access cover 556 can be affixed to thefinger guide 532 with hinges, to allow opening for user loading andunloading of microtip racks 550. The access cover 556 may include a setof indicators associated with each slot that inform the user of thestatus of loaded microtip racks 550. In some embodiments, theseindicators are a set of LEDs, the color of which indicates the presenceand status of loaded microtip racks. In other embodiments, the status ofthe loaded microtip racks 550 can be indicated on a system display aspart of the system's user interface. Alternative embodiments include butare not limited to incandescent lamps, an LCD display, or other suitablevisual indicators

In some embodiments, an XYZ pipettor may be accessible to the microtipstorage unit 120. In some embodiments, the microtip storage unit 120resides near the front of the system to permit an operator to easilyload and unload microtip racks.

In a preferred embodiment microtips, 542 stored in the microtip storageunit 120 are held in microtip racks 550. FIG. 13( e) shows a microtiprack 550 that has a proximal end 562 and a distal end 566. The proximalend 562 may include a handle assembly 564, which provides the user witha gripping point for insertion and removal of the rack. The distal end566 may include a clasp recess 552, which interfaces with the rack clasp554 of the microtip storage unit 120 on insertion of the microtip rack550. The microtip rack 550 may also include a barcode, RFID chip, onewire device, or other devices that convey information related to themicrotip rack 550 to the system. Each microtip rack 550 holds aplurality of microtips 542. In one embodiment, a microtip rack 550 holds161 microtips 542 in a 7×23 matrix. A microtip rack 550 may be formed bysnapping components together (e.g., as in FIG. 13( f)), or they may befriction fit, welded, or glued together.

In order to dissipate static charges accumulated on the microtips 542,portions the microtip rack 550 that contact the microtips 542, may beconstructed of a conductive or antistatic materials, at least in part.Such conductive or antistatic plastics include carbon-filledpolypropylene, polyacetylene, polypurrole, polyaniline, and polymersmixed or treated with antistatic agents such as aliphatic amines,aliphatic amides, quaternary ammonium salts, phosphoric acid esters,polyols, polyol esters, PEDOT:PSS, and polyaniline nanofibers. In someembodiment only tip support 546 is made from conductive or antistaticmaterials. The rack base 538 of the microtip rack 550 is designedenclose the microtips 542 in order to prevent contamination. In oneembodiment, the rack base 538 includes a clasp recess 552 to secure themicrotip rack 550 within the microtip storage unit 120. The rack base538 may also be made of conductive or antistatic materials. Therelationship between the microtip support 546, microtips 542, andmicrotip rack base 538 is also shown in the exploded view of themicrotip rack 550 shown in FIG. 13( f). Microtips 542 may be furtherprotected from contamination by placement of a rack cover 544 over themicrotips 542. The rack cover 544 is attached to the microtip rack 550;in one embodiment, the rack cover 544 is affixed to the upper peripheryof the microtip rack 550 using an adhesive. The rack cover 544 may becomposed of multiple layers, and may be held in place using adhesive.

A microtip rack 550 may serve to store items other than microtips. Suchitems include reaction vessel bases 246, reaction vessel plugs 222,sealed reaction vessels awaiting further processing, and testing devicesfor use in characterizing thermal cycler performance. In someembodiments microtips may be returned to the microtip rack 550 afteruse, for re-use or eventual disposal.

M. Waste Processing: Waste Processing Lane

FIG. 14( a) shows a perspective view of a waste processing laneaccording to an embodiment of the invention.

FIG. 14( b) shows a perspective view of a liquid waste storage assemblyaccording to an embodiment of the invention.

FIG. 14 (c) shows a perspective view of a waste processing lane inassociation with a solid waste container according to an embodiment ofthe invention.

Following processing of a sample, it may be desirable for the system tohave a device for discarding the used assay cartridge 200 and itscontents, along with other consumables, in a manner that minimizes therisk of contamination and that assures the safety of the user. Asexplained above, with reference to FIG. 1( b), at least one of theprocessing lanes 116 can be used for disposal of used assay cartridges200 following processing of the sample. The waste lane 116(c) has accessto or includes tools for the disposal of both solid and liquid waste.

FIG. 14( a) shows an embodiment a waste lane 870 that includes access toan aspiration probe 986, to a solid waste garage 874, and to a wastecartridge carriage 872. Functions of a waste lane 870 can includeremoval of liquids from assay cartridges 200 and disposal of assaycartridges 200. Alternatively, a waste lane 870 may dispose of assaycartridges 200 without prior removal of waste fluids.

As shown in FIG. 14( a), a waste lane 870 may include an aspirationprobe 986 that removes accessible liquid waste from an assay cartridge200. In some embodiments an aspiration probe 986 is disposed above awaste cartridge guide 990 at a fixed position along the lane motionpath. The aspiration probe 986 may be mounted on a probe elevator 988that facilitates vertical movement of the aspiration probe 986. Theupper wall of the waste cartridge guide 990 can include an opening orgap at the fixed position that is in alignment with a probe elevator 988to allow the aspiration probe 986 access to the assay cartridge 200.

Some components of the waste lane 870 may be located within theprocessing area of the system while others may be located elsewhere onthe system, both for design convenience and to minimize the risk ofcontamination. For example, the risk of contamination from wastematerials is reduced by placing a waste container in a compartment thatis at least partially isolated from the portion of the system dedicatedto sample processing and analysis.

In an embodiment shown in FIG. 14( b), components of a waste lane 870include a peristaltic pump 909, a liquid waste container 908, and a fillsensor 907. These are functionally part of the waste lane, but as shownin FIG. 1( d), these may be stored in an enclosed cabinet in the base ofthe system.

In operation, the aspiration probe 986 enters and drains the assaycartridge 200 of waste or residual liquids. The aspiration probe 986 caninclude a hollow tube that is fluidically connected to a peristalticpump 909, which provides suction. Alternatively, suction may providedvia connection to a negative pressure source, such as a vacuum pump. Insome embodiments, the hollow tube of the aspiration probe is springloaded. This arrangement impels the aspiration probe 986 downward untilthe hollow tube either reaches a pre-set vertical stop or collides withthe bottom of an assay cartridge 200 compartment, assuring that allfluid contents are removed while minimizing damage to the aspirationprobe 986. In some embodiments, the hollow tube of the aspiration probe986 is conductive and in communication with a liquid level sensingcircuit. This permits the system to verify that the aspiration probe hascontacted liquid waste and to verify its successful removal. In analternative embodiment, the waste lane 870 may include a millitippipettor 704, and utilize a millitip 220 to transfer waste fluids froman assay cartridge 200.

As shown in FIG. 14( b), the peristaltic pump 909 can drive drainingaction by transferring fluid through the aspiration probe 986 into aliquid waste container 908. In some embodiments, the liquid wastecontainer 908 may be connected to the aspiration probe 986 and include aconnection to a source of negative pressure, thereby avoiding the use ofan active pumping mechanism. The liquid waste container 908 serves tostores waste fluids and connects by tubing to the peristaltic pump 909.In some embodiments, the liquid waste container 908 includes a fillsensor 907 that monitors the level of liquid stored therein. This fillsensor 907 may be any of a number of sensor types, including a floatvalve, a scale to monitor the weight of the liquid waste container 908,or a capacitive sensor. In one embodiment, the fill sensor 907 is athrough-beam optical sensor. Alternatively, the system may estimate thefill level of the liquid waste container 908 by aggregating the knownfill volume of each assay cartridge 200 compartment that has beendrained. The peristaltic pump 909 and liquid waste container 908 arefunctionally part of the waste lane 870 but may reside outside the wastelane. In an alternative embodiment liquid waste may be transferred fromthe assay cartridge 200 to an external drain, avoiding the need to storeliquid waste on the system.

In one embodiment, the waste lane 870 functions by moving the spentassay cartridge 200 so that the aspiration probe 986 drains successivecompartments of the assay cartridge 200, transferring the drained fluidsto a liquid waste container 908. The waste cartridge carriage 816 in thewaste lane 870 may advance to move a compartment to a position under theaspiration probe 986 and causes the probe elevator 988 to lower theaspiration probe into the compartment. The probe elevator 988 lowers theaspiration probe 986 into a compartment to a depth sufficient to reachthe bottom of the deepest compartment. Spring-loading may stop theaspiration probe 986 at the compartment bottom irrespective of theactual depth. This advantageously accommodates tolerance stack up thatmay contribute uncertainties related to compartment depth.Alternatively, the probe elevator 988 may selectively lower theaspiration probe 986 to depths appropriate for specific compartments. Asthe probe elevator 988 lowers the aspiration probe 986, the system maymonitor a liquid level sensor to determine the fill level of thecompartment, and activate a peristaltic pump 909 or other negativepressure source to begin draining once the aspiration probe contactsfluid. Once the aspiration probe assembly 870 drains a compartment, theliquid level sensor may confirm the efficacy of the draining process bysensing the decreased fill level. After draining, the probe elevator 988raises the aspiration probe 986 and the cartridge carriage 816 advancesto reposition assay cartridge 200 so that the aspiration probe isaligned with the next compartment.

As shown in FIG. 14( c), a waste lane 870 can include a solid wasteejector, which serves to dispose of the assay cartridge 200. The solidwaste ejector is aligned with the waste cartridge guide 800 and may bedisposed at the proximal end of the waste cartridge guide. The solidwaste ejector accepts an assay cartridge 200 from the cartridge guide800 and stores it for operator removal. Components of the solid wasteejector may include a waste garage 874 to accept and temporarilyaccommodate an expended assay cartridge during ejection, a waste chute880 to direct the expended assay cartridge so as to avoid jamming, and asolid waste container 882 to retain the expended assay cartridges. Insome embodiments, the waste garage 874 and the waste chute 880 may becombined in a single component. The solid waste container 882 can befunctionally part of the waste lane 116(c), but may reside outside thewaste lane. As shown in FIG. 1( d) solid waste 92 may be stored in awaste cabinet beneath the system. The system may incorporate featuresthat reduce the probability of or minimize the impact of inadvertentrelease of contaminants. The waste cabinet may include ultraviolet lightsources, In one embodiment, the waste cabinet is maintained at negativepressure, with incoming air, outgoing air, or both passing through HEPAfilters. Such a HEPA filter may be mounted in a manifold that directsair flow to or from different parts of the system through differentregions of a single filter. Air pressure may be monitored on both sidesof such a HEPA filter to determine if the HEPA filter needs to bechanged. In some embodiments, the solid waste container may bedisposable. In other embodiments, the solid waste container may bereusable and used in conjunction with a disposable liner. In order tohelp ensure containment of solid waste the system may include a binsensor that monitors the current capacity of the waste bin, allowing thesystem to notify the user when the waste bin requires emptying. In someembodiments, the system includes a waste bin sensor that allows thesystem to notify a user of failure to replace the waste bin in the wastecabinet after emptying.

The waste garage 874 may be an elongated hollow body, which is open atthe end facing the cartridge guide 800 and which is open at the bottomwhere it couples to the waste chute 880. In some embodiments, the wastegarage 874 and the waste chute 880 can be combined into a single partthat is removable for easy cleaning. In some embodiments, the wastecartridge carriage 872 moves the assay cartridge 200 into the wastegarage 874 as the waste lane 870 drains successive assay cartridgecompartments. Once an assay cartridge 200 is fully within the wastegarage 874, the waste cartridge guide 990 no longer provides support; asa result the assay cartridge 200 falls through the open bottom into theconnected waste chute 880. In other embodiments, the assay cartridge 200is moved into the waste garage 874 without removal of waste liquid fromsome or all of the assay cartridge 200 compartments, effectivelycombining liquid and solid waste disposal functions and simplifying theoperation of the system.

The waste chute 880 may be a hollow body forming a channel large enoughto accommodate an assay cartridge 200. The walls of the waste chute 880may turn so that the channel changes direction from substantiallyvertical to an angle downward of and lateral to the direction of thewaste lane 870 motion path. The angled section directs assay cartridges200 dropping through the waste chute 880 laterally into the solid wastecontainer 882 disposed below. This reduces undesirable stacking ofexpended assay cartridges 200 within the solid waste container 882, asassay cartridges so directed are less likely to nest vertically with oneanother. This advantageously prevents assay cartridges 200 from blockingthe waste chute when the waste container is only partially full. Thewaste chute 880 may include a door that, when closed, provides a barrierbetween the solid waste container 882 and the waste lane 870 in order tofurther isolate contaminated waste.

Since an assay cartridge drops vertically once it leaves the cartridgeguide 800, the waste cartridge carriage 816 of the waste lane 870 maynot manipulate the assay cartridge from the normal controlled surface.As noted above, in other processing lanes the propelling feature 303 ofthe cartridge carriage 816 lies within a gap defined by a controlledsurface 248 and a support tab 218 of the assay cartridge 200. In thewaste lane 870 this arrangement may present a risk of snagging as theassay cartridge 200 drops. In a preferred embodiment, this is preventedby having the cartridge carriage 816 push the assay cartridge 200 fromthe distal surface of the support tab 218. In this arrangement thecartridge carriage 816 does not have the capacity to retract an assaycartridge 200 once it is within the waste lane 116(c), and can onlyadvance it. This advantageously reduces the chances of contamination orsystem malfunction due to inadvertent reintroduction of a used assaycartridge 200 into the processing lanes 116 or transfer shuttle 898. Thesystem may further reduce the possibility of snagging by providingsufficient room within the cartridge guide 800 so that a drained assaycartridge does not fully enter the garage. Processing of the next assaycartridge 200 in succession may then push the previous drained assaycartridge fully into the waste garage 874 and down to the waste chute880.

In some embodiments, the system has one or more accessory waste chutesthat direct solid waste to the solid waste container 882. One of theaccessory waste chutes may be accessible by the sample pipettor assembly700, and may include a passive stripping device for removal of the filmpiercer 268 following piercing of the protective film overlying theassay cartridge 200. This passive stripping device can be a rigid,bifurcate assembly that arches vertically, with the central gap alignedwith the travel path of the sample pipettor assembly 700. In such anarrangement, simple lateral movement of the sample pipettor assembly 700allows the passive stripping device to engage the film piercer 268 andgently release it from the pipette mandrel 728. This advantageouslypermits controlled release of the film piercer, which may have a sharpedge, into an accessory waste chute. An accessory waste chute may beaccessible by the XYZ pipettor 1142. In such an embodiment, the XYZpipettor 1142 can be used to dispose of used microtips 542 and usedreaction vessels 221.

N. Transfer Shuttle

FIG. 14( d) shows a perspective view of a transfer shuttle according toan embodiment of the invention.

FIG. 14( e) shows a transfer shuttle aligned with a processing lane.

Processing of assay cartridges 200 across multiple processing lanes 116can include a mechanism for transfer of the assay cartridge betweenlanes. As shown in FIG. 1( c), in some embodiments, assay cartridges 200are transferred between processing lanes 116 using the transfer shuttle118 at a transfer position. Some processing lanes, such as the cartridgeloading lane 116(f), may only use the transfer position to unload anassay cartridge 200. Other processing lanes, such as the waste lane116(c), may use the transfer position only to load or accept an assaycartridge 200. Other processing lanes, such as an amplificationpreparation lane (116 g), an elution lane (116 e), and a wash lane (116b) may both load and unload assay cartridges 200 at the transferposition. In some embodiments, the transfer position of a specificprocessing lane 116 is proximate to the intersection of the transfershuttle 118 motion path with the lane motion path of that processinglane. The transfer shuttle 118 may be moved between lanes by anysuitable means, including, for example, a gantry system, an overheadcrane, a conveyer belt, or a track with drive wheels.

The transfer shuttle 118 moves assay cartridges 200 among processinglanes 116, as discussed above. In an embodiment shown in FIG. 14( d),the transfer shuttle 898 may include a shuttle gantry 908 and a shuttlechannel 892. The shuttle gantry 908 supports the shuttle channel 892 andmoves it among the processing lanes. The shuttle channel 892 may includealignment sensors 894 for detecting alignment flags 900 on the cartridgeguides 816 of the processing lanes, ensuring proper alignment betweenthe shuttle channel and each cartridge guide. Similar alignment flags897 may also be positioned on the cartridge carriage 816 of a processinglane 116. In some embodiments, the alignment sensors 894 are opticalsensors. Alternatively, alignment sensors may be placed on the cartridgeguides 816 of the processing lanes 116 and alignment flags positioned onthe shuttle channel 892.

The shuttle gantry 908 can be a single-axis linear transport disposedperpendicularly to the lane motion path of the processing lanes 116. Insome embodiments, the shuttle gantry 908 includes a linear transportincluding a shuttle track 896 that is attached to the shuttle gantryextends in the direction of travel. The shuttle track 896 may extend thefull length of the desired travel, and incorporate a shuttle drive 890.A variety of drive systems may be suitable for this purpose, includinglead screw and nut, a linear motor, or a pneumatic actuator. In someembodiments, the shuttle drive 890 includes an idler pulley that isattached to the shuttle track 896 near one end of travel and a fixedmotor connected to a drive pulley that is attached to the shuttle tracknear the opposite end of travel. A timing belt may extend between theidler pulley and the drive pulley, and connect to the shuttle gantry908. The distance between the drive pulley and the idler pulley may beadjustable to simplify installation of the timing belt and to permitadjustment of the tension for optimal performance. The shuttle gantry908 may include a track bearing configured to rest on a portion of theshuttle track 896. In this configuration, rotation of the motor drivesthe timing belt through the drive pulley and moves the shuttle channel892 to various positions along the shuttle track 896. Alternatively, thetransfer shuttle 898 may be any structure capable of reaching each ofthe processing lanes such as a rotary transport, a guided tracktransport, an elevator, an XYZ Cartesian transport, or an articulatedarm.

The shuttle channel 892 may be a section of U-shaped channel similar toa portion of the guide channel 862 of a cartridge guide 800. As with thecartridge guide 800, the interior aspect of the lower wall of theshuttle channel 892 may support an assay cartridge horizontal web 228 onone side and the bottom surface of a cartridge flange 906 on other side.The opening or gap in the lower wall allows the wells and vertical web226 of an assay cartridge 200 to extend below the shuttle channel 892. Alinear spring may serve to align and retain an assay cartridge 200within the shuttle channel 892. In some embodiments, as shown in FIG.14( e), the shuttle channel 892 includes tapering or angled lead-infeatures 904. Such lead-in features 904 may serve to compensate forminor misalignments between the shuttle channel 892 and the guidechannel 862 of a processing lane 116, thereby preventing damage to theassay cartridge 200 during transfer and reducing the frequency of systemfailures due to misaligned assay cartridges.

In one example of how the transfer shuttle 898 can function, the shuttlegantry 908 positions the shuttle channel 892 at the transfer position ina first processing lane. The cartridge carriage 816 of the firstprocessing lane then moves to the transfer position to place the assaycartridge 200 in the shuttle channel 892. The shuttle gantry 908 thenrepositions the shuttle channel 892 at the transfer point of a secondprocessing lane. The cartridge carriage 816 of the second processinglane then moves the assay cartridge from the shuttle channel 892 intothe guide channel 862 of the second lane. The cartridge carriage of thesecond processing lane may move to the transfer position prior to thearrival of the shuttle channel in order to simplify transfer of theassay cartridge 200. During transfer the system may control the transfervelocity of the transfer shuttle 898 in order to reduce splashing of thecontents of the assay cartridge 200.

In another example of how the transfer shuttle 898 can function, atransfer shuttle having more than one shuttle channel positions a firstshuttle channel at a transfer position of a first processing lane. Thecartridge carriage of the first processing lane then transfers a firstassay cartridge to a first shuttle channel of the transfer shuttle. Theshuttle gantry then repositions the transfer shuttle, aligning a secondshuttle channel of the transfer shuttle with the transfer position of asecond processing lane. The cartridge carriage of the second processinglane transfers a second assay cartridge to the second shuttle channel ofthe transfer shuttle. The shuttle gantry then repositions the transfershuttle to align the first shuttle channel with the transfer position ofthe second processing lane. The cartridge carriage of the secondprocessing lane then retrieves the first assay cartridge from the firstshuttle channel of the transfer shuttle for processing within the secondprocessing lane. The shuttle gantry then repositions the transfershuttle to transfer the second assay cartridge to another processinglane, which may be the first processing lane. This operation may bereferred to as a cartridge switch. A cartridge switch may occur within asingle operational pitch, which is described in greater detail below. Insome embodiments, the first processing lane is the cartridgepresentation lane. In some embodiments, the second processing lane is awarming lane.

FIG. 14( g) shows another transfer shuttle 898 according to oneembodiment of the invention. In this embodiment, two shuttle channels892 may be coupled to a shuttle gantry 908, so that two cartridges canbe transported simultaneously. In yet other embodiments, three or moreshuttle channels may be present in the transfer shuttle. This embodimentis advantageous, as it can increase productivity as more assaycartridges can be transferred.

O. XYZ Transport Device

FIG. 15( a) shows a perspective view of an XYZ axis transport deviceaccording to an embodiment of the invention.

FIG. 15( b) shows a perspective view of a portion of a Y axis transportdevice.

FIG. 15( c) shows a Z axis elevator for the XYZ axis transport device.

FIG. 15( d) shows an X′ axis transport device.

As shown in FIG. 1( c), an XYZ transport device 40 is positioned toaccess both sample processing and sample analysis portions of thesystem. According to a more specific embodiment of the invention shownin FIG. 15( a), an XYZ transport device 1100 can comprise a number ofindependent motion systems. The first may be an XYZ axis transportapparatus 1132. In one embodiment, the XYZ axis transport apparatus 1132can associated with (e.g., coupled to) a pipetting arm 1136. The XYZaxis transport apparatus 1132 can move in an X direction, a Y direction,or a Z direction. A second independent motion system may be an X′ axistransport device 1134. In one embodiment, the X′ axis transport device1134 is associated with a slide-lock manipulator 1138 that is used toaccess thermal cyclers 1300. Another independent motion system mayinclude an X-axis transport element 1133. It may include a linear track,as well as a drive device for causing the XYZ axis transport apparatus1132 to move in an X direction. Yet another independent motion systemmay include a Y-axis transport element 1131. It may include a lineartrack, as well as a drive device for causing the X-axis transportelement 1133 to move in a Y direction.

The pipetting arm 1136 may move along both X and Y axes along the majorplanes of the system, and (as shown in FIG. 15( c)) may include a pumpcarriage 1140 that can move vertically in the Z axis. The pump carriage1140 may include a microtip pipettor 1142 similar to those utilized insome processing lanes 116 of the system. This pipettor 1142 can be usedto load and shuck microtips 542, pipette reagents between the reagentstorage unit 124 and processing lanes 116, place plugs 222 in the baseof reaction vessels 221, and transfer PCR reaction vessels 221 to andfrom the thermal cycler cell garage 1200. The XYZ transport can includedevices that facilitate processing of reaction vessels, including mixingdevices capable of releasing air bubbles trapped against the interior ofa reaction vessel. Such devices include orbital mixers, ultrasonicdevices, and devices that spin the reaction vessel. In some embodiments,the XYZ transport may include multiple pump carriages, carrying pipettepumps of with different effective volume ranges.

Alternative embodiments of the system may utilize a dedicated device forthe transfer of reaction vessels, reaction vessel plugs, and microtips.Such dedicated devices may include a gripper configured for “pick andplace” of items such as reaction vessels, reaction vessel plugs, andmicrotips,

The XYZ transport device 1100 may also include positional encoders and alinear encoder reader 1104 that provide positional information andfeedback to a controller, as shown in FIG. 15( b). Examples of suchencoders include magnetic linear encoders that may be incorporated intogantries and other supporting structures and encoders that areincorporated directly into drive motors 1112, such as optical rotaryencoders.

To further refine movement and orientation on the system, the pipettor1142 may include a sensing circuit that signals proximity to and contactwith objects or fluids, either through the pipettor 1142 or an extensionof the pipettor such as a disposable microtip 542. Such a sensingcircuit is described in further detail below, and can be responsive toconductive objects or fluids. Other possible sensing mechanisms includeoptical, acoustic, and radio frequency sensors. Examples of conductiveobjects include conductive pipette tips, conductive plugs 222 for PCRreaction vessels, and conductive surfaces on the system itself. Thissensing circuit can provide confirmation of the presence of a conductivepipette tip 542, plug 222, or plugged PCR reaction vessel 221 on thepipettor, and allows the use of known conductive features on the systemfor calibration of the position of the XYZ transport device 1100.

FIG. 15( b) also shows air valves 1106 for controlling air flow topneumatic systems, a home sensor 1110 for indicating a home position forthe pipetting arm 1136, as well as a carriage mount for the pipettingarm 1136.

The XYZ transport device 1100 may include additional independent motionsystems that may include positional encoders, such as an X′ axistransport device 1134 as described above and as shown in FIGS. 15( a)and 15(d). Such independent motion systems may include a slide lockmanipulator 1138, which moves along an X′ axis can be used to manipulatea slidable cover or door that lies within its motion path. In oneembodiment, the sliding cover is a slidable lid of a thermal cyclermodule (see FIGS. 16( j)-16(m)).

A system according to an embodiment of the invention may comprise alinear track, a pipetting arm coupled to the linear track, and anactuator coupled to the linear track and configured to extend away fromthe linear track and retract towards the linear track. In oneembodiment, the actuator can move independently of the pipetting armalong an X′ axis. In accordance embodiments of the invention, the X′axis can be parallel with the long axis of the thermal cycler modulegarage. FIG. 15( d) shows an embodiment where the slide lock manipulator1138 includes a linear actuator 1124 that terminates in a grippingfeature 1142. FIG. 15( d) also shows an X′-axis motor with a rotaryencoder, a rail 1128, and a conduit cover 1122 (e.g., for covering wiresand other conduits). In an alternative embodiment, the actuator is aslide lock manipulator that moves in concert with the pipetting arm andis coupled to the same motion mechanism.

A pneumatic cylinder can cause the actuator 1124 to extend and retractaway from and towards the X-axis transport element 1133. The pneumaticcylinder may extend in any axis that is suitable for its function. Inone embodiment, the pneumatic cylinder extends along the Y axis. In theembodiment described above, this gripping feature 1142 may be in theform of a cylinder, and may reversibly engage the slidable lid of athermal cycler module (or other analytical unit). In such an embodiment,the XYZ transport can include sensors that determine the position ofsuch a slidable lid. Movement of the linear actuator 1124 results allowsthe system to move the slidable lid, thereby opening or closing thethermal cycler module. The linear actuator 1124 can be a pneumaticcylinder, although other mechanisms that provide linear movement such ashydraulic cylinders, linear stepper motors, worm gear drives, timingbelt and pulley assemblies, and solenoids may also be used.

The gripping feature 1142 may be an expanded section of the terminus ofthe linear actuator 1124, the expanded section having sufficient radiusand thin enough section to engage a complementary feature on theslidable lid of the thermal cycler module. In one embodiment, the X′axis transport device 1134 moves the slide lock manipulator 1138 into aposition adjacent to the thermal cycler module. The slide lockmanipulator 1138 then extends the linear actuator 1124 to engage theslidable lid with the gripping feature 1142. The gripping feature 1142may be released from the slidable lid by reversing this operation. Thegripping feature 1124 can have an approximately circular cross-section,with a rounded edge and a thickness that increases towards the center,however other geometries, including polyhedrons, spheroids, conicalsections, and combinations of the shapes are possible. Alternatively thegripping feature 1124 may incorporate two or more extensions that eitherpassively or actively engage a feature on the slidable lid.

The slide lock manipulator can be used in other embodiments of theinvention. For example, the slide lock manipulator can be used in amethod comprising: acquiring a reaction vessel (e.g., reaction vessel221 in FIG. 5( c)) with a pipetting arm (e.g., pipetting arm 1136 inFIG. 15( a)), opening the analytical unit (e.g., the thermal cyclermodule 1300 in FIG. 16( b)) with a slide lock manipulator (e.g., 1138 inFIG. 15( a)), aligning the pipetting arm with the analytical unit, andreleasing the reaction vessel from the pipetting arm. Thus, theparticular XYZ transport device 1100 shown in FIG. 15( a).

P. Sensor System

As noted above, the system can include a sensor system. In somesubassemblies, a secondary controller may be associated with a sensorsystem that includes a sensing circuit that provides feedback to thesystem. In one embodiment, a subassembly that is associated with asensor system is a pipetting device. A sensor system according to anembodiment of the invention may comprise a mandrel (e.g., element 4110in FIG. 15( e)), which may form part of a pipetting device, and asensing circuit configured to determine a characteristic of an extensionelement on the mandrel. The sensing circuit comprises one or more sensorchannels, coupled to a processor (e.g., controller 4600 in FIG. 15( e))configured to determine the characteristic of the extension elementbased on the error signal. The sensing circuit may comprise aphase-locked loop (also known as a PLL), a plurality of sense channels,a processor or controller, and other components.

An exemplary sensing system comprising a sensing circuit (e.g., a liquidlevel sensing circuit) is shown in FIG. 15( e). The sensing circuitaccording to an embodiment of the invention provides a signal thatindicates when a portion of the subassembly contacts or approaches adiscontinuity in permittivity, conductivity or a source ofelectromagnetic (electrostatic) induction. One example of a detectablediscontinuity in permittivity is an air-liquid interface; for thisreason, such a sensing circuit may be referred to as a liquid sensor. Anexample of a detectable discontinuity in conductivity includes a goodconductor physically attaching to a material with higher resistivity.Examples of detectable sources of electromagnetic induction include anyconductive, charge holding elements in relatively close proximity to theportion of the subassembly under discussion. These discontinuities inpermittivity, conductivity or sources of mutual capacitance, eitherindividually or combined, modify the amount of capacitance “seen” by thecircuit which results in a detectable modulation or change in thesignal. For example, a detectable modulation or change can be indicatedas a PLL “error” signal.

In some embodiments, the sensing circuit can be a liquid level sendingcircuit as described in Radio Frequency Liquid Sensor, or RFLS. Oneexample of an RFLS is found in U.S. Pat. No. 4,912,976, which is hereinincorporated by reference in its entirety, for all purposes. Itdescribes a capacitance-based liquid sensing circuit that includes areactive element that forms part of a tuned circuit in avoltage-controlled oscillator. The current embodiment incorporates arelated capacitance-based liquid sensing circuit that includes adistributed reactive element that forms part of a tuned circuit in avoltage-controlled oscillator. The reactive element can be coarselymodeled as a combination of capacitance, resistance and inductanceincluding a dielectric and terminal conductors. The reactive elementneed not be continuously self-contained or localized, but can changeaccording to the application. The properties of the reactive element canbe altered (and consequently detected) by effecting change to any of thebasic constituents, e.g., changes to the dielectric, to terminalconductors or to the mutual capacitance environment.

Changes in the local environment surrounding one terminal of thereactive element amount to a change in the reactive element'sdielectric. When the permittivity of the dielectric changes, thecapacitance sensed by the circuit can be altered resulting in a changein frequency. This change in frequency can be detected by comparison toa fixed frequency reference. Such a change indicates that one of theterminals of the reactive element has, for example, encountered aliquid.

In some embodiments, one of the reactive element terminals is a liquidhandling probe that forms part of the RFLS circuit. Alternatively, oneterminal of the reactive element may be altered adding a conductiveextension of the subassembly that is discarded after use. Examples ofdisposable conductive extension elements include, but are not limitedto, millitips and microtips. In such an embodiment, the sensing circuitcan provide a signal that indicates the successful attachment, andsubsequent release, of a conductive millitip (220 of FIG. 6), microtip(490 of FIG. 12( b)), film piercer (262 in FIG. 4( e)), or reactionvessel plug (222 of FIG. 5) to the pipette mandrel. The sensing circuitcan also be configured to detect different volumes of liquid in apipette tip attached to a mandrel as well as to provide informationregarding the type of liquid.

As an example of changing the mutual capacitance environment, anotherembodiment of the liquid level sensing circuit can be used to detect theapproach of a pipette mandrel (which forms one of the reactive elementterminals) to one or more conductive targets (which can form otherreactive element terminals) that are placed within the path of thepipettor. This approach can be a patterned series of movements thatcomprise a search for a conductive target in 3-dimensional space that isinitiated once the pipette mandrel is brought into proximity to theconductive target. Such information, when combined with informationregarding the position of associated stepper motors, can be used forautomating alignment of the pipettor within the system. The conductivetargets may be fortuitously located system components or conductivetargets incorporated into the system for this purpose. Conductivetargets can include projections that extend from a system component.Examples of projecting conductive targets include substantially planartabs and cylindrical pins. Alternatively, a conductive target can be ahole or gap in an otherwise continuous conductive surface. Anydiscontinuity or array of discontinuities in a conductive element can beused for detection purposes. The detected modulated signal can be usedto measure alignment, proximity, contact, speed, acceleration, directionand vibration in addition to other parameters. This can be useful incharacterizing a range of mechanical performance specifications.

FIG. 15( e) is a simplified block diagram of a sensor system 4000,according to one embodiment of the present invention. The sensor system4000 may be incorporated into or associated with the pipetting arm 1136shown in FIG. 15( a). The sensor system 4000 may be configured toperform multiple functions including liquid level detections, basicinstrument alignment functions, pipette tip (or other device detection;as described above) detections and detections of discontinuities inpermittivity, conductivity, and sources of electromagnetic(electrostatic) induction. The sensor system 4000 includes aphase-locked loop based sensor (“PLL sensor” or “sensing circuit”) 4100,a level sense channel 4200, a first alignment channel 4300, a secondalignment channel 4400, a direct current (“DC”) sense channel 4500, amultiplexor (“mux”) 4550, an analog-to-digital converter (ADC) 4560, adigital-to-analog converter (DAC) 4570, a processor 4600, a memory block4620, a digital potentiometer 4640, and an input-output (“I/O”) portextender 4660, all operatively and/or electrically coupled together. ThePLL sensor 4100 includes a reactive element 4110, a filter and relayblock 4115, an inductive-capacitive-resistive (“LCR”) tank circuit 4120,a voltage-controlled oscillator (“VCO”) 4130, a phase-frequency detector(“PFD”) 4140, a reference oscillator 4150, and a filter 4160. The tankcircuit 4120 includes a first set of varactors 4124 and a second set ofvaractors 4122. The first set of varactors 4124 are connected to the VCO4130 and the reactive element 4110 through the filter and relay block4115. The midpoint of the first set of varactors is connected to theprocessor 3600 through the DAC 4570.

The sensor system 4000 may further comprise a plurality of sensechannels. For example, the sensor system 4000 may comprise a level sensechannel 4200, which includes an amplifier circuit 4210 and a filter4220. It may also include a first alignment channel 4300, which includesan amplifier circuit 4310 and a filter 4320, and a second alignmentchannel 4400, which includes an amplifier circuit 4410 and a buffer4420. It may further include a DC sense channel 4500, which includes anamplifier circuit 4510 and a filter 4520.

The reactive element 4110 may comprise a pipette mandrel, or a pipettemandrel in combination with an extension element such as a pipette tip,piercer, pipette tip with liquid, etc. The reactive element 4110 may beconfigured to sense changes in the surrounding dielectric and sensechanges due to electromagnetic induction. Furthermore, the sensor system4000 may be configured to determine a characteristic of an extensionelement of the pipettor mandrel. For example, the reactive element 4110may include an extension element such as a film piercer or a reactionvessel, each with different electrical properties, and the sensor system4000 can determine if the extension element is present or has changed inany way. Other reactive elements 4110 and extension elements may be usedand would be known and appreciated by one of ordinary skill in the artwith the benefit of this disclosure. In certain embodiments, thereactive element 4110 (e.g., mandrel) can have a resistance, a reactance(e.g., capacitive reactance or inductive reactance), or a combination ofboth (e.g., an impedance).

The PFD 4140 is a multistate phase-frequency detector configured forphase-locked loop applications where a minimum phase and frequencydifference between a reference and a VCO is achieved when the loop islocked. The PFD 4140 is further configured to compare the frequency ofthe VCO 4130 to the frequency of the reference oscillator 4150 (i.e., afixed oscillator) and generate a corresponding difference voltage orerror signal. The error signal is proportional in magnitude anddirection to the difference between the VCO and reference outputfrequencies. As further described below, the PLL error signal (from PLLsensor 4100) can be used as the source of all measurement channels(e.g., level sense channel 4200) in the sensor system 4000. The errorsignal generated by PFD 4140 can be fed back to the VCO 4130 through thefilter 4160, where the VCO 4130 adjusts its operating frequency until itmatches the frequency of the reference oscillator 4150. In this “locked”condition, the error voltage is relatively constant (non-changing) andis continuously monitored by the processor 4600. In one embodiment, thefilter 4160 is an active filter to provide for a wide range of VCO 4130tuning voltages. In some embodiments, the operating frequency of the VCO4130 or the reference oscillator may be multiplied or divided. Inanother embodiment, the VCO 4130 operating frequency is a function ofthe tank circuit 4120, the filter relay block 4115, and the reactiveelement 4110.

The LCR tank circuit 4120 controls the frequency of the VCO 4130 andincludes the reactive element 4110. This reactive element may bedistributed; in one example such a distributed reactive element includesfilter block 4115. When the reactive element 4110 experiences a changein capacitance, the frequency of the LCR tank circuit 4120 also changes.A change in any element of the LCR tank circuit 4120 (i.e., capacitance,resistance, or inductance) causes a change in the frequency of the VCO4130, thus changing the PLL error voltage monitored by the processor4600. Changes in the capacitance of the LCR tank circuit 4120 may becaused by a number of events including pipette tips touching liquid,mandrels passing in close proximity to conductive targets, and theplacement of a pipette tip on a mandrel. The LCR tank circuit 4120includes two sets of varactors, which function as voltage controlledcapacitors. The first set of varactors 4124 is configured to adjust thesensitivity of the system 4000. The sensitivity is changed by makingadjustments to the operating point of the sensor system 4000. Thesensitivity adjustment is performed by altering the point of interplaybetween the first and second sets of varactors, thereby providing forvery sensitive responses for very small changes in capacitance as wellas smaller responses for large changes in capacitance. For example,biasing 4124 at a high capacitance, forces 4122 to a low capacitance dueto the PLL locked condition. Any required change in the capacitance 4122due to the operation of the PLL requires a relatively high voltage dueto the position of the operating point. This results in enhancedsensitivity. Likewise, biasing 4124 at a low capacitance, forces 4122 toa high capacitance. Any required change in the capacitance 4122 due tothe operation of the PLL requires a relatively low voltage due to theposition of the operating point. This results in decreased sensitivity.The first set of varactors 4124 is configured to exploit the shape ofthe varactor characteristic curves to improve sensor performance for awide range of applications. The operation and exploitation of varactorcharacteristic curves to improve the sensitivity of the sensor 4000would be known and appreciated by one of ordinary skill in the art withthe benefit of this disclosure. The second set of varactors 4122 isconfigured to provide a variable voltage input to adjust the VCO 4130output frequency. In an alternative embodiment, the PLL sensor 4100 isconfigured to compare the phase of the VCO 4130 with the phase of thereference oscillator 4150 to generate a corresponding differencevoltage. Such phase comparisons may be made using a voltage phasedetector. In one embodiment, the VCO 4130 is configured to operate at anominal frequency of 6 MHz. In another embodiment, the referenceoscillator 4150 is a crystal oscillator. Further embodiments may includedifferent configurations of the phase/frequency detector, the loopfilter, the charge pump (combined with the loop filter) and the tankcircuit, including additional active or passive devices, which would beappreciated by one of ordinary skill in the art with the benefit of thisdisclosure

The filter and relay block 4115 is configured to filter out bothradiated and received radio-frequency (“RF”) energy, according to anembodiment of the invention. The filter can be tuned to calibrate theresponse of the sensor 4000. Filter and relay block 4115 furtherincludes a relay configured to both remove any potential charge on themandrel and provide a transient startup pulse to the PLL loop if needed.

The filter 4160 is configured as a multi-pole filter or integrator witha charge pump function, according to an embodiment of the invention. Thefilter 4160 is configured to receive the output of the PFD 4140 andgenerate a DC error signal proportional to the difference between thereference oscillator 4150 frequency and the VCO 4130 frequency. Thesteady-state DC error level is fed back to the VCO varactors to maintaina minimum phase and frequency difference with respect to the referenceoscillator output frequency.

The level sense channel 4200 is configured to respond to small PLL errortransients that are induced when a pipette tip enters or exits a liquid,according to an embodiment of the invention. The PLL error signal can beAC-coupled to the programmable gain, single-supply amplifier circuit4210 and through filter 4220. The PLL error signal may be characterizedas an AC-coupled phase-locked loop error voltage, which may be anexample of a first error signal or second error signal. In an embodimentof the invention, the amplifier can be in a non-inverting configuration.In other embodiments, the filter 4220 may be a multiple-stage Sallen-Keylow-pass filter. The output of the level sense channel 4200 is directedto both a fully differential analog interface (e.g., mux 4550) connectedto a remote microcontroller (e.g., processor 4600) ADC and to a localADC 4560. The time constant of this signal chain allows the sensorsystem 4000 to respond to transient entry and exit liquid level senseevents encountered by a conductive pipette tip attached to a conductivemandrel (i.e., reactive element 4110). On entry and exit events, theoutput signal of level sense channel 4200 is configured to produce apositive or negative signal (relative to mid-supply), respectively. Inone embodiment, the level sense channel 4200 output signal is sent tothe processor 4600 for further processing by way of the mux 4550 and/orthe ADC 4560. The processor 4600 is configured to determine, forexample, whether the reactive element 4110 (e.g. a pipette tip) entersor exits a liquid based on the electrical characteristics of the levelsense channel 4200 output signal. To illustrate, the PLL error signaloperates at a nominal value when the pipette tip (e.g., reactive element4110) is not making contact to other objects or mediums. In other words,the PLL error signal can be a substantially constant voltage when thepipette tip is not touching anything. As described above, a positivesignal excursion (e.g., positive voltage “spike”) occurs when thepipette comes into contact with a liquid. The magnitude of the spikedepends on the various electrical characteristics of the liquid. Thelevel sense channel 4200 is configured to detect the positive ornegative voltage spike and thus determine that the pipette has madecontact with the liquid. There are a variety of ways that the levelsense channel 4200 can detect a positive or negative voltage spike. Inone embodiment, the level sense channel 4200 compares the magnitude ofthe voltage spike (i.e., contact with a liquid) to the magnitude of thenominal voltage (i.e., no contact) and measures the difference voltage.The nominal voltage can be referred to as a reference voltage. Inanother embodiment, the level sense channel 4200 may offset the positivereference voltage slightly higher than the nominal value to filter outany small positive voltage spikes that may occur due to noise on thechannel. Similarly, the level sense channel 4200 may offset the negativereference voltage slightly lower than the nominal value to filter outany small negative voltage spikes due to noise on the channel. In someembodiments, the sensor system 400 can store the reference values (i.e.,reference voltages, offset reference voltages, etc.) in memory block4620. Other output signal configurations may be used and would be knownand appreciated by one of ordinary skill in the art with the benefit ofthis disclosure.

In certain embodiments, the level sense channel 4200 can also detect afill level of an extension element (e.g., a pipette tip). For example,certain extension elements can hold a certain volume of a liquid. Theelectrical characteristics of the extension element will changedepending on how much liquid is present inside the extension element. Toillustrate, an extension element with no liquid inside may have acertain reactance which may yield a certain PLL error voltage. Anextension element filled with a liquid may have a different reactanceand thus a different PLL error voltage. The level sense channel 4200 isconfigured to measure and quantify the difference between the twovoltages (including the other detectable water levels and error voltagesin between). It should be noted that this type of measurement isdifferent from detecting an initial contact with a liquid. As describedabove, when the extension element comes into initial contact with aliquid, the level sense channel 4200 measures a voltage spike in the PLLerror voltage relative to a nominal value. In contrast, the level sensechannel 4200 is measuring the change in the nominal value as the amountof water in the extension element changes. For example, slowly adding aliquid to the extension element would cause the PLL error signal (i.e.,the nominal value) to slowly increase or decrease depending on theelectrical characteristics of the liquid. Quantifying and/or calibratingthe level sense channel 4200 to accurately measure a fill level of anextension element based on the changing PLL error signal would beunderstood by those of ordinary skill in the art with the benefit ofthis disclosure. In other embodiments, DC sense channel 4500 may detectthe fill level of an extension element (e.g., a pipette tip) asdescribed in more detail below.

The first alignment channel 4300 is configured to optimize the sensorsystem 4000 response to instrument alignment targets, according to anembodiment of the invention. The amplifier circuit 4310 includes anAC-coupled, single supply, dual stage amplifier with programmable gains.More specifically, the dual stage amplifier includes a high-gain andlow-gain section followed by a filter 4320. The first alignment channel4300 is configured to receive an AC coupled phase-locked loop errorvoltage, which may be an example of a first error signal or second errorsignal. In one embodiment, the filter 4220 performs a simple low-passfunction. The output of the first alignment channel 4300 is directed toboth a fully differential analog interface (e.g., mux 4550) connected toa remote microcontroller (e.g., processor 4600) analog-to-digitalconverter (“ADC”) and to a local ADC 4560. The time constant of thesignal chain allows the sensor system 4000 to respond tohigher-frequency transient alignment events as sensed by the motion of aconductive mandrel (e.g., reactive element 4110) in close proximity to aconductive target. In one non-limiting example, the output signal of thefirst alignment channel 4300 produces a negative going response when amandrel (i.e., reactive element 4110) approaches a conductive target,and a positive going response when the mandrel moves away from thetarget. In one embodiment, the output signal of the first alignmentchannel 4300 is sent to the processor 4600 for further processing by wayof the mux 4550 and/or the ADC 4560. The processor 4600 is configured,in one non-limiting example, to optimize the sensor system 4000 responseto instrument alignment targets based on the electrical characteristicsof the first alignment channel 4400 output signal. In some embodiments,the amplifier circuit 4310 may comprise one or more amplifier stage andmay or may not include the filter 4320. In one embodiment, the amplifiergain is set by the programmable digital potentiometer 4640.

The second alignment channel 4400 is configured to optimize the sensorsystem 4000 response to instrument alignment targets, according to anembodiment of the invention. The amplifier circuit 4410 includes anAC-coupled, single supply, dual stage amplifier with programmable gains.More specifically, the dual stage amplifier includes a high-gain andlow-gain section followed by a filter 4420. In one embodiment, thefilter 4420 performs a simple low-pass function. The output of thesecond alignment channel 4400 is directed to both a fully differentialanalog interface (e.g., mux 4550) connected to a remote microcontroller(e.g., processor 4600) ADC and to a local ADC 4560. The second alignmentchannel 4400 also includes a programmable offset function. The timeconstant of this signal chain allows the sensor system 4000 to respondto lower-frequency transient alignment events as sensed by the motion ofa conductive mandrel (e.g., reactive element 4110) in close proximity toa conductive target. In one non-limiting example, the channel 4400produces a negative going response when a mandrel (i.e., reactiveelement 4110) approaches a conductive target, and a positive goingresponse when the mandrel moves away from the target. In one embodiment,the output signal of the second alignment channel 4400 is sent to theprocessor 4600 for further processing by way of the mux 4550 and/or theADC 4560. The processor 4600 is configured, in one non-limiting example,to optimize the sensor system 4000 response to instrument alignmenttargets based on the electrical characteristics of the second alignmentchannel 4400 output signal. In some embodiments, the amplifier circuit4410 may comprise one or more amplifier stage and may or may not includethe filter 4420. In one embodiment, the amplifier gain is set by theprogrammable digital potentiometer 4640. The gain of the secondalignment channel 4400 may differ from the gain of the first alignmentchannel 4300. In another embodiment, the second alignment channel doesnot include the filter 4420. The gains of each alignment channel aretypically adapted for different targeting applications.

The DC sense channel 4500 is configured to sense liquids, targets, tipsand some environmental conditions, according to an embodiment of theinvention. The channel 4500 is further configured to evaluate and trackthe performance of the sensor system 4000 as it interacts with aplurality of stimuli (e.g., liquid, solid, and gaseous environments,changes in permittivity, etc.). The DC sense channel 4500 includes anamplifier circuit 4510 that comprises a DC-coupled, single-supply,fully-differential amplifier configured to compare the PLL error signalto a programmable reference bias voltage and to amplify the difference,according to one embodiment of the invention. The resulting differencesignal passes through a filter 4520. The DC sense channel 4500 isconfigured to receive a DC coupled phase-locked loop error voltage,which may be an example of a first error signal or second error signal.In one embodiment, the filter 4520 is a low-pass filter. The output ofthe DC sense channel 4500 is directed to both a fully differentialanalog interface (e.g., mux 4550) connected to a remote microcontroller(e.g., processor 4600) ADC and to a local ADC 4560. The time constant ofthe signal chain allows the sensor system 4000 to respond to bothtransient events and steady-state conditions as sensed by the motion orstatic condition of a conductive mandrel or probe (e.g., reactiveelement 4110). Furthermore, the DC sense channel 4500 produces acontinuous DC output signal allowing it to sense effects which aresemi-persistent such as tips installed on a mandrel, the fill level ofan extension element such as a pipette tip, etc. For example, mandreltips may alter the electrical properties of the reactive element 4110thereby causing a shift in the DC channel output voltage. A DC referencebias voltage may be programmed to compensate for such semi-persistentchanges in the PLL error signal. In one embodiment, the output signal ofthe DC sense channel 4500 is sent to the processor 4600 for furtherprocessing by way of the mux 4550 and/or the ADC 4560. The processor4600 is configured, in one non-limiting example, to sense liquids,targets, tips, and environmental conditions based on the electricalcharacteristics of the DC sense channel 4500 output signal. Toillustrate, an extension element on the mandrel with no liquid insidemay have a certain reactance which may yield a certain PLL errorvoltage. DAC 4570 may apply a DC reference bias voltage to compensatefor the reactance of the extension element. Upon filling, an extensionelement filled with a liquid may have a different reactance and thus adifferent PLL error voltage. The DC sense channel 4500 is configured tomeasure and quantify the difference between the two voltages (thereference bias voltage compensating for the PLL error voltageattributable to the extension element alone and the PLL error voltageattributable to the extension element including the filling liquid). Insome embodiments, this may include comparing the output of DC sensechannel 4500 to one or more stored reference values established by acalibration procedure. Quantifying and/or calibrating the DC sensechannel 4500 to accurately measure a fill level of an extension elementbased on the PLL error signal would be understood by those of ordinaryskill in the art with the benefit of this disclosure. Alternatively,other amplifier and filter configurations may be used and would be knownto one of ordinary skill in the art with the benefit of this disclosure.

The DAC 4570 is configured to adjust sensor sensitivity, alignmentchannel offset, DC channel offset, and DC channel reference. In oneembodiment, the DAC 4570 is a 4-channel device. The local ADC 4560 is an8-channel device positioned on the sensor board (not shown) configuredto sample the sensor signals from the various channels previouslydiscussed (e.g., first alignment channel 4300). The ADC 4560 includesadditional functionality to generate interrupts based on sensed eventsto an associated microcontroller or computer, such as processor 4600.

The mux 4550 can comprise two multiplexors configured to direct varioussensor signals (e.g., from the first or second alignment channels) toone of two analog differential buffers. These analog channels areconnected remotely (off of the PCB) to the ADC of the microcontroller(processor 4600).

The processor 4600 handles system communications and signal processingtasks associated with the sensor system 4000 (e.g., implementingpipetting functions). Furthermore, the processor 4600 provides theinterface to the sensor system 4000 for communication and datamanagement tasks, and the processor 4600 can be a remote or localmicrocontroller. The processor 4600 may be configured to receive analogoutputs (e.g., from the level sense channel 4200) through the MUX 4550or from the digital output from the ADC 4560. The processor 4600 isfurther configured to digitize analog signals and provide additionalcontrol functions to the various channels including the second alignmentchannel 4400 and DC sense channel 4500, as shown in FIG. 15( e). In anembodiment, the processor 4600 resides within a same module as thesensor board (not shown).

In one embodiment, the processor 4600 is configured to communicate withthe memory block 4620, digital potentiometer 4640, and I/O port extender4660. The memory block 4620 is configured as local memory for datastorage. The digital potentiometer 4640 is configured to adjustmeasurement channel gains, as described above. The I/O port extender4660 is configured to apply control to the multiplexers, relay block,and phase-frequency detector (connection not shown).

There are a number of advantages to embodiments of the inventionincluding the use of a PLL circuit for detecting an impedance of aprobe. As described above, the impedance of the reactive element 4110(e.g. the probe), and by extension the impedance measured at the tankcircuit 4120, determines the operating frequency of the VCO 4130. Animpedance describes a measure of opposition to alternating current (AC)which comprises a measurement of the relative amplitude and phase of thevoltage (V) and current (I). An impedance typically has a complexelement which can be described as a resistance (R) plus a reactance (X).The reactance may be, for example, a capacitive or inductive reactance.Typically, in order to measure the impedance of the probe, an AC signalsource, a source voltage measurement, and a current measurement isneeded. The current measurement can be transformed into a secondaryvoltage measurement (through I-V conversion) where the resultingmeasurements may be expressed in vector coordinates (magnitude andphase). As such, the PLL sensor 4100 provides a convenient structurewell suited to perform impedance measurements because it can beconfigured to automatically align the phase and perform a single voltagemeasurement to indicate, with high accuracy, any changes to the compleximpedance (R+iX) as required by the sensor system 4000.

Embodiments of the invention can incorporate several features that canresult in successful utilization of the distributed reactive element insensing discontinuities in permittivity, conductivity or sources ofelectromagnetic induction.

1. The sensing circuits can use both AC and DC modes of operation whichenable detection of both transient and steady-state signals. The AC modeis useful when looking only for change or transient events in thepresence of noise such as when entering or exiting a liquid. The DC modeis useful when continuous tracking of conditions is needed. This canoccur for example, when the mandrel is tracked to determine if ittouches or attaches (to varying degrees) to any conductive element.These modes can be applicable over different sensor sensitivities. Forexample, the DC mode is useful in quantizing environmental effects athigh sensitivity while at low sensitivity, the DC mode is useful for tipdetection.2. The sensing circuits can incorporate methods for adjustingsensitivity without adjusting gain. This allows accommodation of a verywide range of sensing applications with the same hardware whileproviding enhanced noise performance. It also allows the hardware toperform outside its normal expected range of application thus extendingits usefulness. This is done through voltage dependent capacitance. Thevoltage dependence of the spacing between charges on the two sides of apn junction can be used to facilitate sensitivity adjustments.3. The realization of one terminal of the reactive element incorporatesshielding in such a way as to enhance noise performance. This imposesspecific lengths on the antenna connection between the sensor and themandrel.4. The inclusion of an electrical switch device in this embodimentallows several additional functions, including discharge of the pipettemandrel and a mechanism for reliable startup of the phased locked loop.

Q. Thermal Cycler Modules

As noted above, PCR or “Polymerase Chain Reaction” refers to a methodused to amplify DNA through repeated cycles of enzymatic replicationfollowed by denaturation of the DNA duplex and formation of new DNAduplexes. Denaturation and renaturation of the DNA duplex may beperformed by altering the temperature of the DNA amplification reactionmixture. Real time PCR refers to a PCR process in which a signal that isrelated to the amount of amplified DNA in the reaction is monitoredduring the amplification process. This signal is often fluorescence;however, other detection methods are possible. In an exemplaryembodiment, a PCR subsystem takes a prepared and sealed reaction vesseland performs a complete real-time polymerase chain reaction analysis,thermal cycling the sample multiple times and reporting the intensity ofemitted fluorescent light at each cycle.

The PCR subsystem can comprise several subsystems including an opticalexcitation subsystem, an optical detection subsystem, a PCR reactionvessel including plug, one or more thermal cycler modules 1300 and athermal cycler garage 1200. The PCR subsystem can be supported by atransport device such as an XYZ transport device.

In embodiments of the invention, a thermal cycle can refer to onecomplete amplification cycle, in which a sample moves through a timeversus temperature profile, also known as a temperature profile, thatincludes: heating the sample to a DNA duplex denaturing temperature,cooling the sample to a DNA annealing temperature, and exciting thesample with an excitation source while monitoring the emittedfluorescence. A typical DNA denaturing temperature can be about 90° C.to 95° C. A typical DNA annealing temperature can be about 60° C. to 70°C. A typical DNA polymerization temperature can be about 68° C. The timerequired to transition between these temperatures is referred to as atemperature ramping time. Ideally, each thermal cycle will amplify atarget sequence of nucleic acid by a factor of two. In practice,however, amplification efficiency is often less than 100%

The system may comprise one or more analytical units. In someembodiments, the analytical units may comprise thermal cycler modules.For example one or more thermal cycler modules can be housed in ahardware structure called a thermal cycler garage, which provides power,communication, and chassis mounts for each. The thermal cycler garagemay house about 20 thermal cycler modules, although the number can varydepending on the throughput requirements of the system.

A reaction vessel can refer to a plastic consumable containing RNA orDNA from a patient sample, target sequence specific primers and probes,a “master mix” that includes nucleotide monomers and enzymes necessaryfor synthesis of new DNA strands, and process control materials. Smallfluid volumes facilitate rapid heat transfer, so the total liquid volumecontained in the reaction vessel is minimal. A typical volume can be 40μL to 50 μL.

In general, the thermal cycler module can: (1) accept a prepared andsealed reaction vessel with sample and reagents, (2) press the vesselinto a temperature controlled thermal block, (3) rapidly cycle the blockand associated sample through a defined temperature profile, (4) exposethe sample to one or more excitation light sources at the appropriateportion of the temperature cycle, and (5) accommodate the opticalcollection path of emitted fluorescence to be sent to the detector.

As shown in further detail below, a thermal cycler module for performingreal time PCR within a PCR reaction vessel can comprise a thermal blockwith a receptacle for receiving a PCR reaction vessel. A slidable lidcan overlap with the thermal block and can have an open position and aclosed position, the slidable lid moving longitudinally between the openand closed positions. It may also include an excitation optics assembly,the excitation optics assembly located beneath the thermal block. It mayfurther include an emission optics assembly, which can be locatedadjacent to the thermal block. The locations of these assemblies can bereversed in some embodiments.

FIG. 16( a) shows a side, perspective view of a thermal cycler module1300. The thermal cycler module 1300 comprises an enclosure 1312 in theform of a rectangular box-like structure. The rectangular, box-likestructure can allow a large number of thermal cycler modules to beplaced in a relatively small area. Although the thermal cycler module1300 is in the form of a box-like structure, it can be in any othersuitable shape or configuration.

An excitation optics assembly 1304 is used to provide excitationradiation to a sample in the thermal cycler module 1300. The emissionsoptics assembly 1302 is used to receive and transmit emissions radiationfrom the sample in the thermal cycler module 1300. Both the excitationoptics assembly 1312 and the emissions optics assembly 1302 aremechanically and operationally coupled to the enclosure 1312.

FIG. 16( b) shows a side, cross-sectional view of a thermal cyclermodule. The enclosure 1312 of the thermal cycler module 1300 may includean enclosure recess 1312(a), which may be cooperatively configured toreceive a slidable lid 1315. The slidable lid 1315 may comprise a body1315(a), which may define a cavity 1341. A biasing element 1344 such asa spring may be attached to an upper portion of the body 1315(a). Acompression head 1342 may be coupled to the biasing element 1344, andmay be perpendicularly oriented with respect to the orientation of thebiasing element 1344. As will be explained in further detail below, thecompression head 1342 can push down on a reaction vessel 221 so that itis in good thermal contact with a thermal block assembly 1311 comprisinga thermal block. An electronics and blower assembly 1313 in the thermalcycler module 1300 can heat and cool the thermal block in the thermalblock assembly 1311, thereby heating and cooling the sample in thereaction vessel 221. When a sample in the reaction vessel 221 isundergoing thermal cycling, light from the excitation optics assembly1304 can provide light to a sample in the reaction vessel 221. Lightemitted from the sample in the reaction vessel 221 can exit the thermalcycler module 1300 through the emissions optics assembly 1302.

FIG. 16( c) shows a garage 1200 with a plurality of thermal cyclermodules 1300. The garage 1200 may comprise a number of linear garagerail structures 1200(a), and adjacent pairs of these garage railstructures 1200(a) may define a garage port 1200(b). The rail structures1200(a) may be in the form of inverted “T” shaped beams, which mayengage side recesses in the thermal cycler module 1300. The garage 1200may hold one, two, three, four, or five or more thermal cycler modules1300. The number of thermal cycler modules 1300 on the system can beoptimized to address throughput needs. In one embodiment, the thermalcycler garage 1200 contains 20 thermal cycler modules 1300. They may bealigned with each other, and may form a compact array. In anotherembodiment, the thermal cycler garage may hold thermal cycler modules1300 in a radial or circular arrangement. The thermal cycler modules1300 may rest on a base 1202, which may have a number of slots 1204formed in it. The slots allow optical cables of excitation opticsassemblies 1304 to pass through.

As noted, the thermal cycler garage 1200 provides power, communications,and chassis mounts that secure the thermal cycler modules 1300 (e.g.,PCR cells) within the system. The number of thermal cycler modules 1300housed in the thermal cycler garage 1200 can be a function of thethroughput requirements for the system. In one embodiment, the thermalcycler garage 1200 houses about 20 thermal cycler modules. The thermalcycler garage 1200 may also incorporate indicators (not shown in FIG.16( c)), such as LEDs, that indicate the status of individual thermalcycler modules 1300. These indicators may provide the user with visualcues, for example color, that signify the current temperature or portionof the temperature profile within the thermal cycler modules 1200. Powerand communications are provided by one or more printed circuit boards.

Referring to FIG. 16( b), the thermal cycler module 1300 may alsoinclude slidable lid 1315 that is normally closed during thermalcycling, but opens to provide access to a thermal block of a thermalblock assembly 1311. In one embodiment, the slidable lid 1315 can be aslide-lock lid that slides at the top of the thermal cycler module 1300,moving parallel to the plane of the system. The motion of a slidable lid1315 can be utilized to perform accessory operations other than closingthe thermal cycler module 1300. Such operation include seating of thereaction vessel 221 within the receptacle of the thermal block assembly1311, releasing the thermal cycler module 1300 from the receptacle ofthermal block assembly 1311, manipulating an optical shutter mechanismthat reduces the amount of ambient light entering the detection opticswhen the sliding lid is open, and providing a fluorescent target thatcan be utilized for alignment of the system's optical subsystem.

FIG. 16( d) shows an optical shutter, which may alternatively bereferred to as a shutter element 1320 that may be incorporated into aslidable lid 1315. It includes a narrow first portion 1320(a), and awider second portion 1320(b) that is integrally formed with the firstportion 1320(a). The narrower first portion 1320(a) is at a middle partof one end of the wider second portion 1320(b). The thermal cyclershutter element 1320 may be made of any suitable material (e.g., metal,plastic, etc.) that may flex and may have resiliency.

FIG. 16( e) shows a perspective view of a portion of a thermal cyclermodule with the shutter element 1320 in a closed position. The firstportion 1320(a) of the shutter element 1320 can be positioned between apair of optical elements, and can be near a thermal block assembly 1311.

FIG. 16( f) shows an internal side view of a portion of a thermal cyclermodule with the shutter element 1320 in a closed position while acorresponding slidable lid 1315 is in an open position. The slidable lidis an example of a movable lid. Other types of movable lids, can move,but need not slide. As shown, the slidable lid 1315 may have an internalrecess 1315(c), which may receive the second portion 1320(b) of theshutter element 1320. A securing element 1321 in the thermal cyclermodule 1300 may secure an end of the second portion 1320(b). As aresult, the narrower first portion 1320(a) lifts up, light can pass fromthe sample in the reaction vessel 221 to the light emission light pipe1401.

FIG. 16( g) shows an internal side view of a portion of a thermal cyclermodule with the shutter element 1320 in a closed position, while thecorresponding slidable lid is in an open position. As shown, a bottomsurface of the slidable lid 1315 pushes down on the second portion1320(b) of the shutter element 1320 so that the first portion 1320(a) ispushed downward. The first portion 1320(a) thereafter blocks any lightfrom entering the emission light pipe 1401. This configurationadvantageously prevents stray light from entering the downstream opticaldetection system (not shown) when the thermal cycler module is open andnot in use

In other embodiments, the shutter element 1320 prevents stray light fromentering the downstream optical detection system when the thermal blockassembly is exposed, rather than when the thermal cycler module 1300 isnot in use. For instance, an open thermal cycler module 1300 may be inuse to temporarily hold a reaction vessel 221 in order to accommodatescheduling of the XYZ pipettor elsewhere on the system.

FIG. 16( h)-1 shows a partial internal perspective view of internalcomponents of a slidable lid 1315. FIG. 16( h)-2 shows a side,perspective view of the slidable lid 1315. The slidable lid 1315 mayinclude a body 1315(a), which may define an elongated aperture 1341 (onehalf of which is shown in FIG. 16( h)-1). The elongated aperture 1341may house a biasing element 1344, which is coupled to a compression head1342. An aperture 1340 for receiving a reaction vessel (not shown) is ata top portion of the slidable lid 1315. The aperture 1340 allows thereaction vessel to pass though the slidable lid 1315.

FIG. 16( i)-1 shows a side, cross-sectional view of a slidable lid 1315in a thermal cycler module, where the slidable lid 1315 is in a closedposition. As shown, a forward portion of the slidable lid 1315 fits intothe enclosure recess 1312(a) of the thermal cycler module enclosure1312. The compression head 1342, impelled by a biasing element 1344,pushes down on the reaction vessel 221 thereby forcing it into a heatblock and providing good thermal contact with the heat block. In oneembodiment, the compression head 1342 is brought into contact with thereaction vessel 221 when the slidable lid 1315 is closed.

FIG. 16( i)-2 shows a side, cross-sectional view of a slidable lid 1315in a thermal cycler module, wherein the slidable lid 1315 is in an openposition. To move the slidable lid 1315 to an option position, it iswithdrawn from the enclosure recess 1312(a). As it is withdrawn, thecompression head 1342 is no longer in contact with the reaction vessel221, and downward pressure is no longer applied. Further, as theslidable lid 1315 is withdrawn, an upwardly tapered ridge pushes up on awider vessel plug third portion 222(c) so that it is pushed upwardthereby disengaging the reaction vessel 221 from the thermal block ofthe thermal block assembly 1311. Because the reaction vessel 221 maypushed down in intimate contact with the thermal block for an extendedtime, it can be difficult to remove from the thermal block after thermalcycling. The design shown in FIG. 16( i)-2 advantageously andefficiently provides for automatic separation of the reaction vessel 221from the thermal block.

The system may utilize an actuator with a gripping feature to open andclose the slidable lid 1315. FIGS. 16( j)-16(k) show a gripping featurethat is configured to manipulate a slidable lid. The gripping feature1350(a) may be part of an XYZ gantry in some embodiments of theinvention, as described above. As shown in these Figures, a grippingfeature 1350(a) can be in a retracted position in FIG. 16( j). In FIG.16( k), the gripping feature 1350(a) is in an extended position and ismanipulated so that it is in between two thermal cycler modules. It thenmoves laterally to engage an end portion of the slidable lid 1315. Asshown in FIGS. 16( l) and 16(m), after it engages the end portion of theslidable lid 1315, it can retract and can also pull the slidable lid1315, thereby separating the slidable lid 1315 from the previouslydescribed enclosure in the thermal cycler assembly.

FIG. 16( n) shows a side, cross-sectional view of an excitation opticsassembly, in position beneath a thermal block 1311(a) in a thermal blockassembly 1311. The thermal block 1311(a) may also define a thermal blockreceptacle, which may contain and be cooperatively structured with thereaction vessel 221. An excitation optics assembly may be locatedbeneath the reaction vessel 221.

More detailed descriptions of operation follow with reference to FIGS.16( a)-16(n). One accessory operation of the slidable lid 1315 can beseating a reaction vessel 221 within a receptacle in a thermal blockassembly 1311 of a thermal cycler module 1300. Thermal transfer isfacilitated by close contact between the thermal block assembly 1311 andthe surface of the reaction vessel 221. The conical shape of thereceptacle of the thermal block assembly 1311 can provide the desiredcontact when a downwards vertical force is applied to an insertedreaction vessel 221.

This downward force can be provided by a slidable lid 1315 thatcomprises a biasing element 1344. The biasing element 1344 can overlapwith the thermal block assembly 1311. It may comprise a segment ofresilient tubing, a spring, a pneumatic cylinder, or other suitabledevice. In one embodiment, the biasing element 1344 can be interposedbetween a curved force director in the form of a compression head 1342,and the inner surface of the top of the slidable lid 1315. As shown inFIG. 16( i), the compression head 1342 and the biasing element 1344 canbe positioned within the slidable lid 1315 so that the apex of the forcedirector is oriented towards the thermal block and is positioned overthe receptacle 221 of the thermal block when the slidable lid 1315 is inthe closed position. In this configuration, the compression head 1342 isimpelled upwards as the slidable lid 1315 closes if a reaction vessel221 is engaged in the receptacle of the thermal block assembly 1311.Resistance from the biasing element 1344 asserts a downwards forceagainst the top of the vessel plug 222 that impels the reaction vessel221 into the receptacle of the thermal block assembly 1311, firmlyseating both the vessel plug 222 in the reaction vessel base 248 and thereaction vessel 221 in the thermal block 1331, and holding the reactionvessel 221 in place during thermal cycling. The amount of force directedagainst the reaction vessel 221 can be five or more pounds, preferablyaround 12 pounds.

The downwards force may be applied by other mechanisms. The slidable lid1315 may include an inclined plane that increases in thicknesslongitudinally, oriented such that the inclined plane contacts andapplies force to the reaction vessel 221 as the slidable lid is closed.The slidable lid 1315 can house a segment of linear spring, positionedto contact and apply a downwards force against the reaction vessel 221.In another embodiment, the slidable lid 1315 can incorporate a linearactuator, positioned to align with the reaction vessel 221 when theslidable lid 1315 is closed.

Firm seating of a reaction vessel 221 within the receptacle of thethermal block assembly 1311 is desirable for optimal heat transfer.However, this practice can lead to difficulty in removal of a reactionvessel 221 after thermal cycling. The motion of the slidable lid 1315can be utilized to ensure that an inserted reaction vessel 221 can bereleased from the thermal block assembly 1311 for transfer elsewhere onthe system. The reaction vessel 221 may, for example, be retrieved usingthe pipettor assembly of the XYZ transport device.

As shown in FIG. 16( i)-1, in one embodiment, a slidable lid 1315includes a lid base plate that lies immediately above the thermal blockassembly 1311. The lid base plate 1347 can comprises an elongatedaperture 1341, the elongated aperture 1341 comprising a proximalterminus, a distal terminus, and parallel edges extending between theproximal terminus and the distal terminus. The distal terminus of theelongated aperture 1341 can be aligned with the receptacle of thethermal block assembly 1311 when the slidable lid 1315 is in the closedposition. The thickness of the lateral edges of the elongated aperture1341 can increase progressively from the distal terminus to the proximalterminus of the elongated aperture 1341 to form a tapered ridge 1346that can engage a top portion of a reaction vessel 221 that is seated inthe receptacle of the thermal block assembly 1311. The lid base plate1347 can be oriented such that this tapered ridge engages and providesan upwards impetus to the inserted reaction vessel 221 as the slidablelid 1315 moves from the closed to the open position. This impetus issufficient to loosen the reaction vessel 221 within the receptacle ofthe thermal block assembly 1311 following thermal cycling, allowing thepipettor assembly of the XYZ transport device to engage and remove thereaction vessel 221 from the thermal cycler cell 1300. A hole 1351 canbe provided in the slidable lid 1315, so that an XYZ transport devicecan retrieve the reaction vessel 221.

As shown in FIGS. 16( f) and 16(g), the slidable lid 1315, when closed,can serve to block exterior light that might interfere with detectionfrom entering the thermal cycler module 1300. When multiple thermalcycler modules 1300 are used there is the further possibility ofexterior light entering through the detection optics of an open thermalcycler module 1300 interfering with measurements being made in adjacent,closed thermal cycler modules 1300. In one embodiment, the thermalcycler module 1300 may further comprise a shutter element 1320, whichmay be a spring shutter. The shutter element 1320 being positioned inproximity to the detection optics assembly of the thermal cycler module1300. The shutter element 1320 is responsive to movement of the slidablelid 1315 and may be resilient. Movement of the slidable lid 1315 to theopen position can displace the shutter element 1320 so that it extendsinto the detection optics assembly, blocking at least a portion of theambient light from entering the detector. Movement of the slidable lid1315 to the closed position can subsequently allow the shutter element1320 to retract from the detection optics assembly, permittingmeasurement of fluorescence from the reaction vessel 221 during thermalcycling.

The slidable lid 1315 can have additional accessory functions that areindependent of its movement. The slidable lid 1315 may include afluorescent target that can be utilized to calibrate the opticalsubassembly of the system, as portions of it may be within range of theemission and detection optics when there is no reaction vessel 221engaged in the receptacle of the thermal block assembly 1311. Thefluorescent target can be a curved force director such as a compressionhead 1342 comprised of a suitable fluorescent material. Alternatively,the entire slidable lid 1315 may comprise a fluorescent material inorder to simplify the manufacturing process. Suitable fluorescentmaterials include fluorescent polymers and structural materials withfluorescent coatings. Also, the slidable lid 1315 may also comprise aheater in some embodiments. Such a heater can be used to preventcondensation from forming within a reaction vessel 221 that is engagedin the receptacle of the thermal block assembly 1311.

FIG. 16( o) shows a side perspective view of a thermal block assembly.FIG. 16( p) shows a top, perspective view of a thermal block assembly1311. As shown therein, the thermal block assembly 1311 can include athermal block 1311(a), a thin film heater 1319 attached to the thermalblock 1311(a), and a lateral aperture 1362. The thermal block 1311(a)may define a receptacle 1311(b) for a reaction vessel (not shown). Thelateral aperture 1362 may allow light to pass from a sample to detectionoptics downstream of the reaction vessel in the thermal block 1311(a).Temperature sensing elements 1364 may be associated with the thermalblock 1311(a). These can be used to measure the temperature of thethermal block or of a reaction vessel held therein. The temperature ofthe reaction vessel or its contents may be determined directly orderived from the temperature of the thermal block. Temperature sensingelements include thermistors and thermal imaging devices.

In embodiments of the invention, the thermal block 1311(a) may compriseany suitable characteristics that support rapid thermal cycling of areaction vessel. For example, it may comprise a substantially planarthermal mass for transferring thermal energy, and a receptacle forforming a thermal contact surface with a vessel. The receptacle cancomprise a frustum of a conical shape and having an upper opening and alower opening, the receptacle being affixed to the front surface of thethermal mass. The thermal block may be composed of a highly thermallyconductive material such as copper, copper alloy, aluminum, aluminumalloy, magnesium, gold, silver, or beryllium. The thermal block may havea thermal conductivity of about 100 W/mK or greater and a specific heatof about 0.30 kJ/kgK or less. In some embodiments, the thermal block hasa thickness between about 0.015 inches and about 0.04 inches. It mayalso have a plurality of heat transfer fins. The thermal block can alsocomprise a heating element that provides the heat that is transferred tothe reaction vessel. The heating element can be a thin film heateraffixed to the back surface of the planar thermal mass, although otherheat sources such as resistance heaters, thermoelectric devices,infrared emitters, streams of heated fluid, or heated fluid containedwithin channels that are in thermal contact with the thermal block mayalso be used. The thermal block may also include one or more temperaturesensors that are used in conjunction with a controller to control thetemperature of the thermal block by, for instance, a PID loop. Thesetemperature sensors may be imbedded in the thermal block. The thermalblock may comprise an optical aperture, where the optical aperture ispositioned to permit optical communication through the planar thermalmass to the interior of the receptacle. This aperture can serve as anoptical window for the detection optics.

The receptacle of the thermal block 1311(a) may also have any suitablecharacteristics necessary to secure the reaction vessel and ensure goodthermal contact with it. For example, in some embodiments, the walls ofthe conical receptacle 1311(b) have an angle of about 1 degree to about10 degrees, an angle of about 4 degrees to about 8 degrees, or an angleof about 6 degrees. The decreasing internal radius of the receptacleensures that as the reaction vessel that is pressed into the receptacleof the thermal block the exterior of the reaction vessel is brought intointimate contact with the interior of the receptacle. The receptacle ofthe thermal block 1311(a) may also have an upper opening and a loweropening. The upper opening allows for insertion of the reaction vessel.The lower opening allows for reaction vessels to fit tightly within thereceptacle 1311(b) despite variation in the length of the vessel thatcan be a consequence of the manufacturing process. The lower opening mayalso act as an optical window for the excitation optics. The thermalcycler module 1300 can include containment features, such as O-ringseals or containment vessels that encompass all or part of the thermalblock, to reduce the risk of contamination from reaction vessels held inthe receptacle 1311(b).

The thermal cycler module 1300 may also include any suitable opticalcomponents. Excitation optics may include an optical fiber in opticalcommunication with a light source and a lens that directs light emittedfrom a terminus of the excitation optical fiber into a reaction vesselengaged in the receptacle of the thermal block. Alternatively,excitation light may be provided by a light source that is incorporatedinto the thermal cycler module and is in optical communication with areaction vessel engaged in the receptacle of thermal block without anintervening optical fiber. Suitable light sources include but are notrestricted to lasers, LEDs, and other high output light sources. LEDsused for excitation may emit an essentially single wavelength or emitmultiple wavelengths in order to simulate white light. Multiple singlecolor LEDs may be used to provide excitation light at differentfrequencies. Detection optics may include an optical fiber that is inoptical communication with a detector located elsewhere on the systemand a lens that directs light emitted from a reaction vessel engaged inthe receptacle of the thermal block into a terminus of the detectionoptical fiber. Detection optical fibers from multiple thermal cyclermodules may be directed to a single detector. Alternatively, detectionoptical fibers may be associated with individual detectors associatedwith specific thermal cycler modules. In another embodiment, thedetector may be mounted within the housing of the thermal cycler moduleand placed in optical communication with a reaction vessel engaged inthe receptacle of the thermal block without an intervening opticalfiber. Suitable detectors include, but are not limited to 1D CCDs, 2DCCDs, photomultiplier tubes, photodiodes, avalanche photodiodes, andsilicon photomultipliers. Detectors may also include interferencefilters, diffraction gratings, or similar devices for separation ofemitted light into discrete wavelengths. Detection optics may alsoinclude a shutter mechanism that blocks light from entering the detectorwhen the interior of the thermal cycler module is exposed.

Embodiments of the invention may also include optical casings forexcitation and emission optics assemblies. These optical casings serveto protect lenses, optical filters, and waveguides associated with theexcitation and emission optics. The optical casing may also includefeatures that facilitate mounting and alignment of the excitation andemission optics in the thermal cycler. An optical casing can have acircumferential groove in the outer surface. Such a circumferentialgroove permits an optical casing to be held in place with a latchingmechanism incorporated into the thermal cycler. In one embodiment, thelatching mechanism is a spring-loaded latch. In such an embodiment, theuser can press the spring loaded latch of the thermal cycler to removeor install an optical casing. The optical casing may be rotationallysymmetrical, so that they are not orientation specific. In oneembodiment, the excitation optical casing is a cylindrical body thatincorporates lenses, optical filters, and waveguides associated with theexcitation optics, having a circumferential groove that interfaces witha latching mechanism of the thermal cycler, and the emission opticalcasing is a cylindrical body that incorporates lenses, optical filters,and waveguides associated with the emission optics, having acircumferential groove that interfaces with a latching mechanism of thethermal cycler. Use of such optical casings simplifies replacement ofoptical components and permit cleaning of the lenses without disassemblyof the thermal cycle.

FIG. 16( q) shows a thermal cycler spring latch that holds an emissionoptics assembly 1357 in the thermal cycler module, as well as anexcitation optics assembly 1359 that is held in place with an excitationspring latch 1339. The spring latches 1337, 1339 can be compressed,thereby removing any biasing force against the emission and excitationoptics assemblies 1357, 1359 and allowing them to be easily removed by auser.

More specifically, the spring latches 1337, 1339 may each comprise abase 1337(b), 1339(b) that is integrally formed with a head 1337(c),1339(c). A biasing element 1337(a), 1339(a) such as a spring may pushagainst the base 1337(b), 1339(b) to bias the head 1337(c), 1339(c) intoa groove (or other type of recess) 1357(a), 1359(a) in the emission orexcitation optics assembly 1357, 1359. To remove the optics assemblies1357, 1359, a user may simply press down on the bases 1337(b), 1339(b)thereby withdrawing the heads 1337(c), 1339(c) from the grooves 1357(a),1359(a), so that they are disengaged from the thermal cycler module1300.

The thermal cycler module 1300 may also include one or more addressablememory units, where the addressable memory unit stores information(e.g., optical alignment information) that is specific for the thermalcycler module. The memory units can be I2C memory blocks, each of whichwith a capacity of about 32 kbits. Individual memory blocks can havedifferent functions. For example, one memory block may be writeprotected memory used to store the serial number and manufacturing testdata specific for that thermal cycler module, where a different memoryblock may have read/write memory that is used to store thermal cyclermodule calibration information, temperature overshoot and undershootinformation, and information related to the number of performance cyclesof various components within that thermal cycler module. Typicalperformance cycles may be the number of heater cycles, the number ofblower cycles, and the total number of thermal cycles completed.

There are also a number of alternative configurations of the thermalblock assembly. In one embodiment, a thermal block assembly holds thereaction vessel near a heating device at one end and has an extendedcooling “tail” region (with or without fins) for use in conjunction witha blower for cooling. In another embodiment, the thermal block assemblycould hold the reaction vessels at one terminus and have an extendedtail with a thin film heater on one side and cooling fins on the otherside, with a blower to direct cooling air to the non-heated side. Otherembodiments can include a cylindrical thermal block, with a centralcavity to hold the reaction vessel, a helical arrangement of coolingfins on the outer surface, and a helical resistive heater nestledagainst the surface of the cylinder that is exposed between the coolingfins. In yet another embodiment, the thermal block may be replaced by anarray of resistive heating wires that surround the reaction vessel,heating it primarily by radiation and convection.

While blowers directing a stream of air may be used for cooling athermal block, in other embodiments of the invention, cooling can beprovided by a heat pipe that is integrated into the thermal blockassembly and is in thermal communication with a large heat sink and fanassembly located elsewhere on the system. Other embodiments of theinvention may include the use of a relatively large thermal mass that ismoved (via pneumatic cylinder, rotary motor, solenoid, linear actuator,mechanical linkage, or other suitable means) into physical contact withthe thermal block to provide rapid cooling. Other embodiments of theinvention can include forced/pressurized air stream could be used inplace of a blower for cooling.

R. Thermal Cycler Module Control

FIG. 17( a) shows a schematic block diagram illustrating some componentsof a thermal cycler module 2100 according to an embodiment of theinvention. Thermal cycler module 2100 may include a power supply 2105.In some embodiments, a power supply is external to the thermal cyclermodule 2100.

Power supply 2105 is connected to thermal block assembly 2110. Thermalblock assembly 2110 may include components (e.g., a heater) that mayprovide heat. A cooling device 2112, such as a fan, may also be coupledto the power supply 2105. An exemplary thermal block assembly 1311 hasbeen described in connection with FIG. 16( b). Thermal block assembly2110 and/or the cooling device 2112 may operate in a binary fashion(being either on or off/heating or cooling) or in a continuous fashion,whereby different applied voltages result in different degrees ofeffective heating and cooling.

Voltage output by power supply 2105 may be at least partly controlled bya voltage signal received from, e.g., an internal processor and internalmemory 2115 and/or an external source (e.g., the signal beingtransmitted via wireless receiver 2120). In one embodiment, the internalmemory includes pre-determined (e.g., testing) voltage signals, whichmay be transmitted to power supply 2105. In one embodiment, a voltagesignal is received from an external source (e.g., an external computersystem). In one embodiment, an initial signal (e.g., a voltage signal ora temperature signal) is received from a source (e.g., by fromtemperature measuring component 2135 a), and a processor in theprocessor and memory unit 2115 converts the initial received signal intoa new voltage signal, which is then sent to power supply 2105. Thefrequency at which temperature data is gathered may be optimized forthermal cycling requirements. A temperature measuring component 2135 amay obtain measurements at intervals ranging from 100 milliseconds to500 milliseconds. In one embodiment, the temperature measuring component2135 a obtains measurements at intervals of about 200 milliseconds.

Thermal block assembly 2110 may be connected to reaction vessel 2125,e.g., to heat and cool the vessel upon receiving a voltage from powersupply 2105. A sample 2130 may be placed within reaction vessel 2125.

Thermal cycler module 2100 may include one or moretemperature-measurement components 2135(a) (e.g., a thermistor).Temperature-measuring components 2135(a) may measure a temperature,e.g., within reaction vessel 2125 and/or thermal block assembly 2110, toproduce a time-dependent temperature signal. Temperature-measurementcomponents 2135(a) may send measured temperature signals to theprocessor and memory unit 2115. In some embodiments a temperaturemeasurement component may send data to an external source, for use incharacterizing the thermal cycler module 2100.

In some embodiments of the invention, the processor and memory unit 2115may comprise one or more microprocessors, coupled to one or more memorydevices (e.g., computer readable media). These devices may be on thesame circuit board, or may be distant from each other, but operativelycoupled to each other. The memory unit may store algorithms forprocessing samples, as well as calibration information. The calibrationinformation may include values specific for the individual thermalcycler module or may include values common to all of the thermal cyclermodules. Calibration information can include factors for calculating thetemperature of the interior of the PCR vessel from the temperature ofthe thermal block.

Each thermal cycler module (e.g., within a thermal cycler garage) may beinfluenced by various environmental or hardware factors affecting theprecise temperature profile that it will exhibit in response to adefined voltage. One factor that may influence a thermal cycler module'stemperature is the ambient temperature. FIG. 17( b), for example, showstemperature measurements of a heat block and a sample, in response to anapplied voltage signal, when the ambient temperature was either 36° C.or 22° C. At the lower ambient temperature, the sample and blocktemperature ramping times were faster, leading to faster cycle times.Within a garage, the ambient temperature of a thermal cycler may beaffected by its relative location. For example, thermal cyclerspositioned close to a perimeter of the garage may sit at a lower ambienttemperature as compared to more centrally located thermal cyclers. Thus,thermal cycles in a garage may gradually experience phase shiftsrelative to each other.

Another factor that may influence a thermal cycler module's temperatureprofile is the thermal cycler module's hardware components. For example,slight variations within each cycler's thermal block assembly (e.g.,including a fan and a heater) may cause variable temperature profilesacross cyclers. FIG. 17( b) shows temperature profiles of a thermalblock and a sample for two different cyclers. Though the cyclers includethe same hardware components, minor differences in the hardware mayaccount for the observed difference in ramping and cycle times.

The temperature-profile variation among thermal cyclers may lead toinconsistent rates of DNA amplification across the cyclers. Thus, DNAamplification may be inconsistent across days (e.g., based onvariability of a garage-surrounding temperature) and even across cyclerswithin a single amplification session. Additionally, the phase shiftscaused by variable temperature profiles may make it difficult to obtainreliable fluorescent measurements of amplification. In embodiments ofthe invention, since a reaction vessel may be assigned to any thermalcycler, variation in performance between different thermal cyclers maycontribute to overall variation in assay performance. This negativelyimpacts system precision and, potentially, both the ultimate sensitivityof an assay and the accuracy of the final reported results.

In one embodiment, control of thermal cycler performance is achievedusing a PID (proportional integral derivative controller) control loop.The thermal block is fitted with thermistors that give temperatureinformation. A typical thermal cycle may shift the temperature of thethermal block between about 70° C. and about 95° C. To achieve a thermalblock temperature of 70° C., a fixed voltage is applied until thattemperature is achieved. Temperature is then maintained using a PIDcontrol loop and temperature data from the thermal block. To raise thetemperature of the thermal block to 95° C., a fixed voltage is againapplied until the desired temperature was reached. Similarly, to reducethe temperature a fixed voltage may be applied to a blower that directsair over the thermal block. The air supplied to this blower may be atambient temperature or may be chilled. Other cooling methods, such asthe use of a directed stream of pressurized air, flow of a cooling fluidthrough channels in the thermal block, and the use of Peltier coolingdevices in thermal contact with the thermal block may also be used.Since it is desirable to minimize cycle times voltages may be selectedthat minimize heating and cooling times generating the fastest possibletemperature ramping rates achievable by each thermal cycler.

In another embodiment of the invention, an algorithm, which can bestored in the memory unit of the processor and memory unit 2115, can beused to produce identical temperature versus time profiles across allthermal cycler modules. Such an algorithm compensates for sources ofvariation in the temperature ramping rates of different thermal cyclers.Such an algorithm may also compensate for different environmentalconditions. Sources of variation can include ambient temperature (FIG.17( b)), thermal block performance, and blower performance (hardwarevariation; FIG. 17( c)).

FIG. 17( d) shows a flowchart illustrating a method according to anembodiment of the invention. In the method, a plurality of thermalcycler modules may be provided as described above (block 2005). Theplurality of thermal cycler modules may be 2, 3, 5, 6, or 7 or more.

Then, a thermal cycler module may be selected (block 2010). The selectedthermal cycler module may be one of many thermal cycler modules. Theother thermal cycler modules that are not selected may form a set ofthermal cycler modules. A set of thermal cycler modules may comprise 1,2, or 3 or more thermal cycler modules.

The thermal cycler module may be selected in any suitable manner. It canbe selected as the least responsive thermal cycler module in the arrayof thermal cycler modules. For example, the selected thermal cyclermodule may be the slowest ramping thermal cycler module in the array ofthermal cycler modules. It may correspond to a longest cycle time or theslowest heat transfer of a thermal cycler module. In other embodiments,the temperature vs. time profile does not need to be based on the leastresponsive thermal cycler module, but can be based on the performance ofa different type of thermal cycler performance characteristic.Regardless of how the temperature vs. time profile is created, theseembodiments of the invention can address both overshoot and individualthermal cycler performance issues.

After the thermal cycler module is selected, a temperature vs. timeprofile is created for the selected thermal cycler module profile (block2015). It can then be stored in a memory unit (e.g., a computer readablemedium such as a memory chip) in the processor and memory unit 2115.

After the temperature vs. time profile for the selected thermal cycleris created, the thermal block assembly of each thermal cycler module inthe array can be adjusted using a source of variation (e.g., ambienttemperature) and the predetermined temperature vs. time profile (block2020). This can be done by obtaining the predetermined temperature vs.time profile associated with a selected thermal cycler module in anarray of thermal cycler modules. The array of thermal cycler modules cancomprise the selected thermal cycler module and a set of thermal cyclermodules. A processor in the processor and memory unit 2115 then controlsthe thermal cycler modules in the set of thermal cycler modules so thattheir performance matches the predetermined temperature vs. timeprofile. Each of the thermal cycler modules in the set of thermal cyclermodules can be controlled using a source of variation between thethermal cycler modules in the array.

Illustratively, the least responsive thermal cycler module that givesacceptable performance in a plurality of thermal cycler modules can beselected. A temperature vs. time profile can then be created using theselected thermal cycler module. An algorithm is then created, and isused to control the thermal block assembly 2110 (and hence the heatprovided by to the reaction vessel) as well as the cooling device 2112.The algorithm uses the selected temperature vs. time profile, andinformation about a source of variation such as the ambient temperatureof the thermal cycler module to determine how to control the thermalblock assembly 2112 and the cooling device 2112. The following equationcan be used in the algorithm:

dB/dt=h _(a) +k(Ta−B(t)),  (1):

where

dB/dt=change in thermal block temperature in degrees per second;

T_(a)=ambient temperature (° C.);

h_(a)=thin film heater output at ambient temperature (° C./second);

k=rate of heat transfer; and

B(t)=the temperature of the thermal block at a given time t.

If B(t) is not measured directly, one can integrate and solve for B(t)to get the temperature of the thermal block at any given time t:

B(t)=(B(0)−(h _(a) /k)−Ta)e ^(kt)+(h _(a) /k)+Ta,

where

B(0)=starting block temperature at time=0.

The processor in the processor and memory unit 2115 can control the thinfilm heater output (h_(a)) by applying modulated pulses of voltage tothe thermal block assembly 2110, and can control the rate of heattransfer (k) in a similar fashion using the cooling device 2112 (e.g.,blower, a fan, or cooling fluid). Alternate methods for modulatingheater and fan output are also possible in embodiments of the invention.

In equation (1) above, dB/dt at a given time can be determined from thepredetermined time vs. temperature profile of the selected thermalcycler module, and the ambient temperature of the thermal cycler module,Ta, can be measured by a temperature measuring component (e.g., athermistor). The variables h_(a) and k can be controlled independently,and both can be varied simultaneously (i.e. the heater and the blowercan be used in combination) to satisfy equation (1).

FIG. 17( e) shows an example of block temperature measurements from 20independent thermal cycler modules that were programmed using theabove-described algorithm. As shown in FIG. 17( e), the thermal cyclermodules in the array of thermal cycler modules perform consistently.This can be advantageously done without significant hardwaremodifications or requiring narrow product specifications. Use ofconsistent thermal profiles among all thermal cyclers on the systemadvantageously reduces variation in the PCR process due to hardwaredifferences and environmental factors. Use of consistent thermalprofiles also produces identical thermal cycling times in every thermalcycler on the system, permitting an accurate estimate of when thermalcycling will be complete for a given sample and simplifying resourcescheduling.

Q. Optics Systems

Embodiments of the invention can also include an excitation anddetection subsystem (herein called detection subsystem). The detectionsubsystem can be responsible for exciting the dyes in the assay andquantifying the fluorescence emitted at each PCR cycle. Both excitationand emission can occur over a range of wavelengths. Light used to excitethe fluorescent dyes can, for example, range from 400 nm to 800 nm. Thedetector used to measure light emitted from the dyes can, for example,be sensitive to light ranging from 400 nm to 800 nm. The detectionsubsystem includes hardware and software components from the lightsource(s) through to the detection on the CCD camera. This includes allthe optical components with each thermal cycler module, the fiber opticsrouting from each thermal cycler module and the spectrophotometermounted under the PCR base plate. The dynamic range of the detectionsubsystem can allow for the detection of amplified PCR products over atleast 3 thermal cycles that are within the linear detectable range ofthe amplification curve or having a range of fluorescence intensity of 2orders of magnitude. The detection subsystem can detect a plurality ofemitted wavelengths from the reaction vessel and to perform thedetection asynchronously across multiple reaction vessels. In oneembodiment up to 7 different dyes can be detected asynchronously amongup to 20 different reaction vessels.

The detection subsystem comprises at least the following components: anexcitation light source, an assembly or assemblies for directingexcitation light to the reaction vessels, an assembly or assemblies fordirecting light emitted by fluorescence occurring within the reactionvessels to a detector, and one or more detectors for measuring theemitted light. The excitation light source can be one or more lasersthat are optically coupled to an excitation fiber optic assembly. Insome embodiments, light from two lasers (for example a 640 nm laser anda 532 nm laser) is passed through line filters to remove light that isoutside of the nominal wavelength range. The beams can be made collinear(or slightly non-collinear). Beams can be made collinear by a variety ofoptical devices, including a beam splitter. In another embodiment, theexcitation laser beams are not made collinear in order to avoidcrosstalk between them. The excitation laser beams can be directed toindividual excitation optical fibers using mirrors mounted in a two axisgalvanometer. Each excitation optical fiber would then direct theexcitation light to an individual thermal cycler module. In oneembodiment, an assembly of 20 excitation optical fibers would be used tosupply excitation light to each of 20 thermal cycler modules. Additionaloptical fibers that are utilized for other purposes may be present inthe assembly of excitation optical fibers; such uses can include opticalalignment. The excitation optical fibers can be held in an orderedarray, with a two axis galvanometer directing light to the input end ofeach excitation optical fiber as needed. In addition, the two axisgalvanometer may direct excitation light to a neutral position where itdoes not enter an optical fiber. Alternatively, an optical switch may beused to direct light from an excitation source to the optical fibers. Avariety of optical fibers are suitable for this use. In one embodiment,the excitation optical fibers are about 200 μm in diameter, and may bebundled in a 4×5 array. In some embodiments, excitation and emissionoptical fiber bundles can include 22 (or more) fibers. Excitationoptical fibers carrying the excitation light terminate in the excitationoptics assembly of the thermal cycler module, which is described above.

Although lasers are the preferred light sources in embodiments of theinvention, embodiments of the invention may include other light sourcesincluding, but not limited to, tunable lasers, individual singlewavelength LEDs, assemblies of single wavelength LEDs, andmulti-wavelength LEDs, white LEDs with a multibandpass filter, and anassembly of single wavelength LEDs and a multibandpass filter.Excitation light sources may be incorporated into the excitation opticsassemblies.

Light emitted from the reaction vessel as a result of exposure to theexcitation light is collected by the emission optics assembly of thethermal cycler module, which is described above. In one embodiment, thisdirects the emitted light to the input end of an emission optical fiber,which subsequently directs emitted light to a detector. In order toimprove coupling efficiency the emission optics assembly may focus theemitted light over an area that is smaller than that of the input end ofthe emission optical fiber. For example, the emitted light may befocused as a 200 μm spot on an emission optical fiber input end havingan area of 800 μm. An emission optical fiber may taper to a smallerdiameter beginning from the input end in order to improve couplingefficiency. Other features, including lenses integrated into the inputend of an emission optical fiber, can be used to increase couplingefficiency. Suitable lens configurations include ball or sphericallenses, aspherical lenses, and graded index lenses.

The detector can be a spectrometer. The spectrometer may be amulti-channel or an imaging spectrometer, which can permit simultaneousreading of multiple optical fibers and reduce the need for switching.The spectrometer can include a multi-bandpass filter between the outputterminus of the emission optical fibers and the detector to selectivelyremove excitation wavelengths. If a single detector is used the emissionoptical fibers may be arranged in a bundle at the input of the detector.Such a multi-channel spectrometer may use a CCD for detection of emittedlight. For example, 20 emission optical fibers from individual thermalcycler modules can be arranged in a 2×10 bundle at the input of adetector. In an alternative embodiment, the detector may be a singlephotodiode, photomultiplier, channel photomultiplier, or similar deviceequipped with an appropriate optical filter. Such an appropriate opticalfilter can be a set of optical filters or a tunable filter.

If a single detector is used the detection system may be able to supportasynchronous measurement of fluorescence from each of the thermal cyclermodules. One way to accomplish this is to use a spectrometer that has anintegration time that is short when compared to the point in the thermalcycle where the read event is to occur. For example, to read during aphase of the thermal cycle that lasts approximately 15 seconds, aspectrometer capable of making an accurate measurement within 50 msec isdesirable. The annealing phase of the thermal cycle, which typicallytakes place at around 60° C., may be used to take advantage of improveddye fluorescence characteristics at lower temperatures. Excitation lightcan be directed to the input end of a specific excitation fiber for therequired integration time using mirrors mounted in a two axisgalvanometer, then directed to another position. If a CCD-based detectoris used the CCD may be cleared between each read event. The CCD may beactivated prior to directing the excitation light to the appropriateexcitation optical fiber and kept active following switching of theexcitation light to a different position in order to facilitate this.The read event can be triggered by monitoring the temperature of thethermal block of the thermal cycler module to ensure that the contentsof the PCR reaction vessel are at the desired temperature. In oneembodiment, the read event can be triggered within a defined portion ofa temperature versus time profile that is applied to a thermal cycler,as described above.

As the throughput of a system increases the complexity of schedulingappropriate read times on a single detector for multiple analyticalunits, such as thermal cyclers, that work in parallel also increases.The workflow for the system, which is described in detail below, mayadvantageously simplify this task by preparing samples for reading in aserial fashion. This ensures that each sample enters the analyticalportion of the system at a different time point, greatly reducing theprobability that a significant number of samples will require that aread event be performed within the same time interval.

FIG. 18( a) shows a detection optics functional block diagram. FIG. 18(a) shows a plurality of light sources comprising a first light source1522 and a second light source 1524, which can provide light to atwo-axis galvanometer and excitation bundle 1520. A control board 1508can provide control signals to the first light source 1522 and thesecond light source 1524. In one embodiment, the first light source 1522may comprise a 640 nm laser, while the second light source 1524 maycomprise a 530 nm laser. However, the first and second light sources1522, 1524 can provide light of any suitable wavelengths.

The two-axis galvanometer and excitation bundle 1520 can receive lightfrom the first and second light sources 1522, 1524, and can becontrolled by a trigger circuit and delay 1514. Light is provided to oneor more reaction vessels as the light passes through the thermal block.Block 1518 depicts excitation and the subsequent emission offluorescence generated from one or more reaction vessels.

Fluorescence radiation from the reaction vessels in block 1518 may becaptured by a collection fiber optic bundle 1516, which may pass theradiation to a spectrometer 1510. In addition to the collection fiberoptic bundle 1516, access to the spectrometer may also be supplied formaintenance and decontamination of the spectrometer 1512, environmentalcontrols that maintain the spectrometer within acceptable operatingconditions 1502, electrical power 1504, and communications with thesystem 1506. The trigger and circuit delay 1514 may be in operativecommunication with the spectrometer 1510.

FIG. 18( b) shows a more detailed diagram of an optics detection systemaccording to an embodiment of the invention. The system comprises acomputer 1616, which can provide control signals to first and secondpower supply and controllers 1610, 1612. The first power supply andcontroller 1610 can supply power to a first light source 1606, while thesecond power supply and controller 1612 may provide power to a secondlight source 1620. Light from the first light source 1608 may passthrough an excitation filter 1606 and may be reflected by an aluminumfront surface coated mirror. Light from the second light source 1620 maybe reflected using a beam splitter. The light beams from the first andsecond light sources 1608, 1620 can then be collinear and can be focusedby a planoconvex lens 1624, reflected by a two-axis galvanometer mirror1626, and directed into an excitation fiber bundle 1644 connected to afiber bundle mount 1620. In other embodiments, the light beams need notbe collinear and may be angled to prevent cross-talk between them.Galiliean telescopes can be used for collimation of the output of bothlight sources (e.g., lasers) and to reduce spot size for coupling toexcitation fibers. Further, initial alignment of the light sources(e.g., lasers) can be performed manually, aligning the beams to a targetholes via coarse adjusters prior to performing automated calibration ofthe galvometric mirror.

In some embodiment, the excitation fiber bundle can comprise twenty (ortwenty two) 200 μm core diameter fibers (CeramOptec, p/n Optran WF,NA=0.12) arranged in 5×4 array with 0.425 mm spacing between the fibers(CeramOptec, p/n RSSLSMA20/20XWF200/220P12/BPGS+BPVC/1.5M/BC).

Exemplary Fiber Bundle Specifications are as Follows:

20 CeramOptec optical fibers (part number WF200/220/245P12, availablefrom CeramOptec of East Longmeadow, Minn.) with the followingspecifications:

a. Pure fused silica core diameter: 200 μm±2%

b. Dopped silica clad: 220 μm±2%

c. Polyimide coating: 245 μm±2%

d. Low OH version

e. Numerical aperture: 0.12±0.02

Light from the excitation fiber bundle can then pass to an excitationlens 1634 and to a reaction vessel 1630 containing a sample via a firstslit 1632. Fluorescent radiation from the sample in the reaction vessel1630 can then pass through a second slit 1636. Once the emissionradiation passes through the second slit 1636, it is focused by acollection lens 1640 and to a collection fiber bundle 1642. Thecollection fiber bundle 1642 is coupled to a spectrometer 1618, whichreceives the fluorescent radiation. Suitable control electronics 1614may be coupled to the computer 1616 and the spectrometer 1618.

FIG. 18( c) shows a perspective view of a detection optics assemblyaccording to an embodiment of the invention. FIG. 18( c) shows adetector in the form of a spectrophotometer 1701 with a 2D array mountedon a plate 1708. A first light source 1703 in the form of a 640 nmlaser, a second light source in the form of a 532 nm laser 1702, mountoptics 1710, and a galvanometer 1705 are also mounted on the plate 1708.Various finned heat sinks 1709, 1711 may also be mounted on the plate1708. An enclosure 1712 in the form of a cover can cover at least thefirst light source 1703, the second light source 1702, the mount optics1710, and the galvanometer 1705. An excitation fiber bundle 1704 can bein operative communication with the first and second light sources 1701,1702.

In one embodiment, the detection optics assembly is supplied as adiscrete, essentially closed unit in order to facilitate fieldreplacement and service. Such a detection optics assembly can includealignment targets in the form of holes that extend through the casing ofthe unit, correct alignment of the light sources encased thereinindicated by observation of light transmitted through an alignmenttarget hole. A detection optics assembly may include coarse adjustmentdevices that extend through the casing of the unit to permit alignmentwithout the necessity of opening the unit. In some embodiments a finalalignment of the light sources is performed in an automated fashionusing a galvanometer mirror.

As noted above, embodiments of the invention can use galvanometers.Alignment can be an issue with 2D galvanometer systems. Embodiments ofthe invention provide each thermal cycler module with a fluorescenttarget that can be observed by the system's detection optics when thereaction vessel is not in the thermal block. In some embodiments, thisis a discrete device shaped like a reaction vessel that is eitherfluorescent or contains fluorescent materials. This is placed in thethermal block of a thermal cycler module for the purpose of aligning theoptics for that module, and can be removed before the thermal cyclermodule is used for PCR. In other embodiments, all or part of theslidable lid are fluorescent, emitting brightly enough to reflect somelight off of the interior walls of the receptacle and into thecollection optics. The opaque plug of the reaction vessel blocks lightto and from the slidable lid during PCR. This provides a fluorescenttarget that sends light down the emission fiber associated with aparticular thermal cycler module when the 2D galvanometer is lined upproperly. To align the optics with a specific thermal cycler, thegalvanometer scans the beam across the excitation fibers while recordingthe position of the galvanometer. When the system identifies theposition that gives maximum intensity from the collection fibercorresponding to the specific thermal cycler module, it records it asthe calibrated position for that thermal cycler. The reason this isdesirable is because, while the use of the galvanometer lets one use acentralized light source and switch back and forth among the differentthermal cyclers very quickly, alignment has to be close to optimal toget good performance. Having an automatable alignment mechanism in placereduces maintenance (manual alignment of 20+ fibers is labor intensive)and provides consistent performance over time.

A similar process may be performed following alignment and prior toperforming thermal cycling in order to assure that the optical path to athermal cycler is not blocked. A significant reduction intensity of thelight observed by the detector in the absence of a reaction vessel,relative to that observed in a prior alignment observation, couldindicate an interruption in the optical path associated with a thermalcycler. The controller may then take actions such as selecting adifferent thermal cycler for the determination that is in process andnotifying the user of a possible fault condition.

Other embodiments of the invention may utilize a single detector forthermal cycler. Such a detector may be an individual spectrometer thatis in communication with each thermal cycler. In another embodiment, thedetector could be a photodiode, photomultiplier, channelphotomultiplier, or similar device associated with each thermal cycler.

S. System Operations and Sample Handling

Many different processing embodiments have been described above, and aredescribed in further detail below.

One embodiment of the invention is directed to a method comprisingloading a sample into a system, and loading an assay cartridge into apreparation location. The assay cartridge includes a reaction well and acompartment. A reaction vessel is in the compartment. The method alsoincludes extracting the nucleic acid in the reaction well, transferringthe extracted nucleic acid from the reaction well to the reactionvessel, moving the reaction vessel to the thermal cycler module, anddetecting the nucleic acid in the thermal cycler module. These and othersteps are described in further detail below.

FIG. 19 shows a flowchart processing methods according to embodiments ofthe invention.

The system according to an embodiment of the invention can be designedto function in a conventional clinical laboratory environment andrequire minimal user intervention. FIG. 19 shows an embodiment wherenormal user interaction with the system is limited to loading of samplesfor analysis 1804, removing remaining samples once they are processed bythe system 1812, replenishing consumables (1814, 1836, 1840), andremoving waste (1828, 1850). In another embodiment, the system is usedin conjunction with an automated laboratory system, and the normal userinteraction with the system is limited to replenishment of consumablesand removal of waste. Other user interactions that are not shown includeperiodic maintenance. This advantageously places a minimal burden placedon the user, in terms of both hands on time and training, which in turnfacilitates integration of the system into the workflow of aconventional clinical laboratory.

A typical workflow for analysis of a sample by the system can bedescribed with reference to the flowchart shown in FIG. 19, withperiodic reference to previously described system components.

Analysis begins by loading a sample onto the system 1802. Samples aregenerally provided in sample tubes, and may be whole blood, serum,plasma, saliva, urine, cerebrospinal fluid, suspensions of fecalmaterial, swabs taken from wounds or other body surfaces, or otherclinically relevant fluids or suspensions. Swabs samples can be providedas tubes containing at least a portion of the swab, with the samplecollection portion of the swab immersed in liquid. Sample tubes may haveindicia that provide identification of individual tubes. Such indiciamay be machine readable, and include one and two dimensional barcodes.

In some embodiments, sample tubes are loaded onto the system by placingthem in a sample holder (block 1802), which can provide support for oneor more sample tubes while providing features that facilitate handling.An exemplary sample holder 616 can be found in FIG. 2( a). The sampleholder 616 may have indicia that provide identification of an individualsample holder 616. Such indicia may be machine readable, and include oneand two dimensional barcodes.

Once a sample tube has been placed in a sample holder 616, it is loadedonto the system by placing the sample holder 616 into an input queue(block 1804). FIG. 1( c) shows an embodiment where the input queue 628is located in a sample presentation unit 110. Various embodiments andfeatures of a sample presentation unit 110 are detailed above. Thesample tube progresses through the input queue 628 until it reaches asample presentation area, where it is identified (block 1806) andbrought into the sample processing workflow. In some embodiments, thesample presentation area is a portion of the sample presentation unit110 and is accessible to a sample pipettor 70. For example, in someembodiments, the sample presentation area may include the presentationtrack 624 shown in FIG. 2( a). Samples may be identified based on theindicia of individual sample tubes, and by their position on anidentified sample holder 616. In some embodiments, the user may manuallydesignate a specific sample via a keyboard 104, as shown in FIG. 1( c),or by other suitable means. Identification of individual samples permitsassociation of the sample with a specific patient, which in turnprovides the system with information regarding the tests to be performedon the sample. Identification of individual samples also allows thesystem to associate results from those tests with an individual patient.

At the sample presentation area, in a sample presentation process (block1808), a portion or aliquot of the sample may be taken from the sampletube for analysis by the system. For example, referring to FIG. 4( a)-1,aliquots may be removed from the sample tube using the millitip 220provided with the assay cartridge 200, and then transferred into areaction well in the assay cartridge 200. This millitip 220 may bereturned to the assay cartridge 200 following aliquot transfer for lateruse.

The system may take one or multiple aliquots from a single sample tubein order to support the performance of multiple tests. When multiplealiquots are taken, the system may first determine the level of fluid inthe sample tube, calculate the volumes required for testing asappropriate for the specified tests, and alert the user if the volume ofthe sample is insufficient to complete all tests. Under suchcircumstances, the system may optimize the order in which aliquots areremoved in order to perform as many tests as possible, or may removealiquots based on a test priority. Alternatively, performance ofmultiple tests may require loading of individual sample tubes for eachtest.

Once aliquot removal from the sample tube is complete, the sample ismoved to an output queue 640 (block 1810). If the sample is held in asample holder, transfer to the output queue 640 may be delayed untilaliquots are taken from all sample tubes in the holder. The output queue640 may be located on a portion of the sample presentation unit 110 (seeFIG. 2( a)), which is described in detail above. Once samples are in theoutput queue 640, they may be removed from the system (block 1812). Theuser may then choose to store the sample tube for possible retesting ofremaining sample or may simply discard the sample tube. Sample may bestored in a sample holder or removed from the holder for more spaceefficient storage.

As described above, sample aliquots are processed by the system usingconsumables. This reduces the probability of contamination due tocarryover. In a preferred embodiment, referring to FIG. 4( a)-1, initialsample processing is performed in a disposable assay cartridge 200.These are supplied to the system by the user, who may place them in acartridge loading unit for temporary storage 1814 prior to use by thesystem. An exemplary cartridge loading unit 112 is shown in FIG. 1( c).The user may place assay cartridges 200 in a cartridge loading unit 112individually, or they may simultaneously place multiple assay cartridges200 in the cartridge loading unit 112. In one embodiment, the lineararrangement of the assay cartridge 200 simplifies the simultaneousgrasping of multiple units, and assay cartridges may be supplied inpackaging with spacing that facilitates this. As noted above, differenttypes of assay cartridge 200 may be utilized. Under these circumstances,different types of assay cartridges 200 may be placed in different areasof the cartridge loading unit 112 for selective introduction into thesystem workflow as they are needed. In the embodiment shown in FIG. 7(a), different types of assay cartridges 200 may be loaded into separatelanes (112(b) and 112(c)). Alternatively, different assay cartridgetypes may carry indicia signifying the cartridge type and may be loadedat any available location in a cartridge loading unit 112 or equivalentstructure. Use of different types of assay cartridges supports the useof different processing protocols, which in turn allows the system toboth process a broader range of sample types and to perform a greatervariety of assays than could be supported by a single type of assaycartridge.

In some embodiments, an assay cartridge 200 is transferred from acartridge loading unit 112 prior to receiving the sample aliquot. Asshown in FIG. 1( b), the assay cartridge 200 can be transferred from thecartridge loading unit 112 by moving the assay cartridge to thecartridge loading lane 116(f). Once in the cartridge loading lane116(f), the assay cartridge 200 may be brought into a position where thesample pipettor 70 can transfer the sample aliquot (block 1816).

In an embodiment of the invention, and referring to FIG. 4( a)-1, theassay cartridge 200 can be supplied with a protective barrier film 205overlying the reagent wells 204, 208, 209. This film 205 can be removedor pierced to gain access to the contents of the reagent wells 204, 208,209. In one embodiment, the system utilizes a piercing element end266(a) of a film piercer 262, shown in FIG. 4( f), to pierce the filmoverlying the reagent wells 204, 208, 209. This film piercer 262 may beconveniently supplied as part of the assay cartridge 200. Film piercingmay take place while the cartridge is in the sample aliquot transferlocation, utilizing the sample pipettor 70 to manipulate the filmpiercer 262. The film piercer 262 may be used prior to transfer of thesample aliquot to the assay cartridge 200, followed by disposal of thefilm piercer 262. The film piercer 262 may have a cutting edge thatslices through the film covering the reagent wells 204, 208, 209 withminimal resistance, thereby avoiding the aerosolization of the wellcontents and subsequent contamination issues. In an alternativeembodiment, the system may utilize the millitip 220 supplied on theassay cartridge 200 to pierce the film covering the reagent wells 204,208, 209, and supply reagents to the reaction well in the assaycartridge.

The assay cartridge 200 can also receive reagents from other sources,which may be stored in a reagent storage unit 124 of the system as shownin FIG. 1( b), while in the cartridge loading lane. Such sources mayinclude bulk bottles. In some embodiments, this is accomplished usingthe XYZ transport device 130. FIG. 9( a) illustrates an embodiment inwhich such reagents are stored in a disposable multiuse reagent pack400. As noted above, the reagent pack 400 contains liquid reagentsrequired for the performance of a specific assay. Examples of materialstransferred to an assay cartridge 200 from a reagent pack 400 at thispoint in the process may include, but are not limited to, processcontrol materials that can indicate successful extraction of nucleicacids, enzymes that support lysis of bacteria, and magneticallyresponsive microparticle suspensions. In some embodiments, materialsfrom the reagent packs are added to the assay cartridge after the samplealiquot has been added. In other embodiments, materials from the reagentpack 400 can be added to the assay cartridge 200 before the samplealiquot is added. In yet another embodiment, some materials from thereagent pack 400 are added to the assay cartridge 200 (e.g., to thereaction well) before the sample aliquot is added while others are addedafterwards.

As noted above, the reagent pack 400 can be a consumable item. Reagentpacks 400 may added to the system by the user via loading (block 1836)into a reagent storage unit 124. An exemplary reagent storage unit 124is shown in more detail in FIGS. 8( a)-8(c). In operation, a user mayrequest that the instrument provide a loading opportunity. In preparingfor the loading opportunity, the system may release selected reagentpacks 400 from the reagent storage unit 10 by releasing the latchassemblies 144 associated with the selected reagent packs. During theloading opportunity, a user may open the RSU access door 126 and viewstatus indicators 140 associated with each loaded reagent pack 400. Theuser may remove any released reagent packs 400 and insert any newreagent packs 400. The instrument verifies the changes by reading theelectronic memory associated with each loaded reagent pack 426. Thereagent pack 400 may hold sufficient reagent for a number of assays, andmay be accessed multiple times while stored within the reagent storageunit 124. During the reaction storage unit operation (block 1838), thesystem may monitor fluid levels within the reagent pack 400 using afluid level sensing circuit in order to determine when the reagent packis exhausted. Alternatively, the system may aggregate data related tothe usage of a reagent pack 400 and relate that data to known fillvolumes in order to determine when a reagent pack is exhausted. Thesystem may notify the user of exhausted or soon to be exhausted reagentpacks so that they can be replaced without impacting workflow (block1844). In some embodiments, the user may remove a reagent pack onrequest for off-board storage.

Following addition of a sample aliquot and any necessary reagents fromthe reagent pack 400, the assay cartridge 200 is transferred to aprocessing area (block 1818). Referring additionally to FIGS. 1( b) and4(a)-1, the assay cartridge 200 is moved from the cartridge transferlane 116(f) to the transfer shuttle 50. The transfer shuttle 50 shuttlemoves the assay cartridge 200 through a series of the processing lanes116 as directed by the protocol associated with the aliquoted sample. Aprotocol may designate the repeated use of a specific processing lane atdifferent times as the protocol progresses. The system may subject assaycartridges to different processing protocols to extract and purifynucleic acids. For example, the system can treat DNA assay cartridgesdifferently from RNA assay cartridges to reflect the physical-chemistryrequirements of the different purification procedures. Further, thesystem may also use different protocols for samples that use the sametype of assay cartridge. For example, DNA extraction from gram positivebacteria may require a different collection of steps to lyse the morerobust walls of the bacteria than the steps required for other DNAisolation. The system may, for example, apply heat to a DNA assaycartridge applied to extraction and purification of DNA from grampositive bacteria. This heating step produces an extended elevatedtemperature that aids in lysis of the gram positive bacterial cellwalls.

The system benefits from applying different protocols by savings in timeand by avoidance of incompatible conditions. Different protocols savetime by skipping unneeded steps. For example, extraction andpurification of DNA from gram positive bacteria requires a period ofheating that is not required for DNA from other sources. While applyinga heating step to such sample may not be harmful, by deleting theheating step the system can process DNA from these other samples morerapidly. This flexibility in processing reduces time to result comparedto the alternative of subjecting all samples to the same timeline.Without use of different protocols the slowest method required by anyindividual assay would necessarily dictate system processing time.

Applying different protocols may avoid incompatible conditions insituations where the conditions for one extraction and purificationprocess are irreconcilable with those of another. A system might adapt asingle processing protocol and avoid some incompatibilities, such asthat due to the gram positive bacteria heating step mentioned above, by,for example, simply placing an assay cartridge in the appropriateprocessing lane without activating the heater. Similarly, false reagenttransfers (i.e. performed without reagent pickup or delivery) ortransfers of inert reagents could possibly allow a common processingprotocol for all samples. Such adaptive methods, however, still limitthe performance of a single processing protocol system performance tothat of the most restrictive method. Further, a common processingprotocol may simply not be possible when mere delay causes theincompatibility. Time delay alone may be problematic, for example, whena protocol depends on the action of an enzyme and the length of timecontrols the extent of enzymatic action. Applying different processingprotocols avoids this processing bottleneck and retains flexibility toapply new or updated methods.

The system applies multiple protocols by routing each assay cartridgethrough a series of processing lanes 116. Each processing lane 116 actson the assay cartridge 200 to perform a subset of the total processingsteps in a protocol. Any given protocol may route assay cartridges 200through some or all of the processing lanes 116. Different protocols mayuse some of the same processing lanes 116. In one embodiment, eachinstance of a protocol routes the assay cartridge 200 associated withthat instance through the same sequence of processing lanes 116 on thesame relative timeline.

Each processing lane 116 may accommodate only one assay cartridge 200 ata time. This advantageously simplifies system design by allowing use ofa single mechanism for transferring assay cartridges 200 betweenprocessing lanes 116 and increases processing flexibility by eliminatingresource conflicts within a processing lane.

Each instance of a protocol may use a consistent pathway and consistenttiming. In this embodiment, for a given protocol each specificprocessing step uses a designated mechanism in a designated location ata designated time relative to the start of that instance of theprotocol. For example, one version of the DNA gram positive isolationand purification protocol requires a transfer of diluent to the reactionwell following addition of magnetically responsive microparticles. Inthis protocol, the transfer can always occur in processing lane 2,always using the processing lane 2 pipettor 244 seconds after the startof sample aliquoting. This practice advantageously reduces assayvariation by assuring that each assay receives the same treatment by thesame mechanisms. Replicates of a single mechanism, even though productsof the same design using the same manufacturing process, may not performidentically. Each replicate suffers variations caused by deviationswithin manufacturing tolerances, local nonuniformities in operatingenvironment, wear and operating history, and from other sources beyondreasonable enumeration.

In one embodiment, the system avoids much of the effect of non-identicalmechanism performance by always using a designated mechanism for eachparticular step in each protocol. This design reduces the need totightly match mechanism performance across different operativelocations. For example, the processing lane 2 pipettor may transfer adifferent actual amount than does the processing lane 3 pipettor withthe same nominal transfer volume. Processing lane 2 may have a slightlyhigher temperature in the vicinity of its pipettor than does processinglane 3. But because each instance of a protocol uses the same pipettorfor a particular operation, the differences contribute an overall biasor systematic error rather than a random error. Such systematicvariations may be corrected through calibration, but random variationsassociated with different mechanisms are much more difficult to correct.The system thus gains the benefits of improved assay precision withoutthe expense and complexity of tightly matched components.

Assay precision may also be improved by reducing the impact of ambienttemperature on sample processing operations. In one embodiment, this isachieved by routing all assay cartridges through a processing lane thatincorporates an assay cartridge heater as an initial process step.Bringing the assay cartridge and its contents to a controlledtemperature prior to the performance of temperature-sensitive processingsteps improves the consistency of the results of such steps as ambienttemperatures fluctuate. The temperature of the assay cartridge and itscontents may be maintained subsequently by the use of assay cartridgewarmers in other processing lanes.

The system may retain each assay cartridge 200 within a particularprocessing lane 116 for a fixed duration. This duration may be the samefor any assay cartridge 200 in any processing lane 116 regardless of theprotocol. This assures consistent timing for all steps in the protocol.Flexible lane-based processing ideally requires transfer of an assaycartridge from any lane to any other lane. In practice, some transfersmay never occur. For example, assay cartridges 200 generally enter theamplification preparation lane 116(g) as shown in FIG. 1( b) only nearthe end of the process, and assay cartridges 200 that enter the wastelane 116(c) may only proceed to the solid waste disposable.

In some embodiments, the system transfers assay cartridges 200 betweenprocessing lanes 116 using a single transfer shuttle 50 in a randomaccess arrangement that permits the transfer of an assay cartridge 200from any processing lane to any other processing lane. The transfershuttle 50 interacts only with the source and destination lane withoutinterfering with any other lane. In one embodiment, the transfer shuttle50 may transfer only one assay cartridge 200 at a time. In this contexttransfer between lanes includes unloading of an assay cartridge 200 fromone identified lane and subsequent loading of the assay cartridge 200into another identified lane. Transfer among processing lanes 116 is abroader term that includes transfer between identified processing lanes116 and also includes the general process of unloading and loadingwithout limitation to particular processing lanes 116. The transfershuttle 50 may have multiple positions for carrying assay cartridges. Inone embodiment, the transfer shuttle 50 includes two or more cartridgeslots 50(a), 50(b). This arrangement permits the exchange of one assaycartridge 200 for another within a processing lane in a single step.This arrangement may allow cartridges to be switched between differentlanes within a single operational, or pitch, interval, as describedbelow. Two or more of such switching steps may be combined to exchangeassay cartridges 200 between processing lanes.

FIG. 20( h) shows a top plan view of a system with two cartridge slots50(a) and 50(b) that can be used for switching assay cartridges 200between different processing lanes 116. The embodiment of the instrumentin FIG. 20( h) includes many other lanes discussed in more detail above.The number and precise configurations and properties of wash lanes116(a) and 116(a)′ (and 116(b), which is not shown in FIG. 20( g)) andtemperature stabilization lanes 116(j) (and 116(h), which is not shownin FIG. 20( g)) may vary based on design and biological objectives.

FIG. 20( i) shows an embodiment of a cartridge-switching process. Atblock 3605, a first cartridge enters a cartridge loading lane 116(f). Atblock 3610, one or more samples and assay process controls are loadedinto the first cartridge, which may be performed in one or more steps.The assay process controls may include a process control compositionused to assess whether later-performed extraction and purification stepswere properly performed. If a control was not sufficiently amplified, itmay be concluded that the samples in the assay cartridge did not undergoproper processing.

At block 3615, a first slot (“Slot A”) 50 a of the transfer shuttle 50engages the first cartridge. At block 3620, the first cartridge is movedby the transfer shuttle 50 to the heating lane 3116(i) and it isunloaded in the lane. The first cartridge may be warmed for a warminginterval, e.g., between about 10-300 seconds, such as about 53 seconds.The first cartridge may be heated to a temperature of about 35-45° C.(e.g., the target temperature is 35° C. plus or minus 3° C.). One ormore of the first cartridge, contents of the first cartridge's mediumwells, contents of the first cartridge's large wells, and contents ofthe first cartridge's reaction vessel component holders may be heated toone or more desired temperatures.

As shown on the right hand side of FIG. 20( i), a second cartridge canbe undergoing a similar set of steps, except that it is behind in time.That is, steps 3705, 3710, and 3715 are similar to steps 3605, 3610, and3615.

At block 3625, a second slot (“Slot B”) 50 b of the transfer shuttle 50engages the warmed first assay cartridge, followed immediately byunloading of the second assay cartridge in Slot A into the heating lane3116(i). This substantially simultaneous transfer of assay cartridgesinto the out of Slots A and B improves the speed of processing, ascompared to the case where there is only one slot in the transfershuttle.

At block 3630, the first cartridge is moved by the transfer shuttle 50back to the loading lane 116(f). At block 3635, reagents are added tothe first cartridge in the loading lane 116(f). At block 3640, the firstcartridge continues to the next lane in a processing recipe. Blocks3725, 3730, 3735, and 3740 are similar to blocks 3625, 3630, 3635, and3640.

As illustrated above, the multiple cartridge slots 50 a, 50 b in thetransfer shuttle 50 may allow for multiple cartridges 200 to be swappedwithin a single lane, or even between adjacent lanes.

In other embodiments, the slots of the transfer shuttle may permit twocartridges to be simultaneously loaded or heated, but not overlappingwithin each other in the loading lane 116(f) or the heating lane3116(i). Thus, a cartridge heater may be at least partly loaded andheated within a single pitch (e.g., about 100-200 s). While the timebetween other processing steps may be approximately the duration of onepitch, both heating and partial or full loading may be occurring withinthe same time interval. This may improve the temporal efficiency of theinstrument. Additionally, by using a two-slot transfer shuttle, a singlemotor may control the movement of both assay cartridges.

In some embodiments, protocols may diverge further from pipelinearchitecture. That is, some protocols including relatively rapidprocessing may start later but finish earlier than other protocolsincluding less rapid processing. This has the benefit of providingfurther flexibility to support rapid protocols without significantconstraint by slower protocols.

The capability for later started assay cartridges to “pass” earlierstarted assay cartridges is available through the flexible capacity ofthe transfer shuttle. The transfer shuttle 50, as described above, maytransfer an assay cartridge 200 from any source lane to any destinationlane; it is not limited to transfers between adjacent lanes. Sincetransfer windows are staggered, the system may, for example, launch afirst protocol routing a first assay cartridge in successive pitches toeach of lanes 1-13 in succession. The system may then launch a secondassay cartridge in lane 1 after the first assay cartridge transfers fromlane 1 to lane 2. The second assay cartridge may in the next pitchinterval transfer from lane 1 to lane 13 where it would complete itsprocessing. Long distance transfers of this type may occur in what wouldotherwise be transfer shuttle idle time. Thus, in such embodiments,later started assay cartridges may finish processing before some earlierstarted assay cartridges. This advantageously allows rapid processing ofselected specimens.

In some embodiments, protocols may include conditional branches. Thatis, the system can process an assay cartridge 200 in a manner wherefurther processing includes a first set of steps if a condition isfulfilled and a second set of step if a condition is not fulfilled. Forexample, the system might transfer an assay cartridge 200 to a wastelane 116(c) if some essential component were missing. In someembodiments, the system might repeat a wash step if washing weredetermined to be inadequate.

Conditions may include anomaly sensing, efficacy sensing, externalinput, or a variety of other conditions limited only by the value ofaltering a protocol on the occurrence of the condition.

Anomaly sensing can include detection of anomalous events such asfailure to detect pick up of a millitip 220, microtip 490, reactionvessel plug 222, or reaction vessel 221. Other examples of anomalousevents include detection of pressure that does not match an expectedprofile or value during pipetting and detection of reagent or samplefill volumes outside of expected bounds.

Efficacy testing can include any test of an intermediate result duringprocessing. For example, the system may assess wash efficacy bymeasuring the amount of residual fluid after washing using the liquidlevel sensor to determine the height of fluid in the reaction well 202.Other exemplary efficacy tests include measurement of assay cartridgetemperature after exposure to a lane heater 1103 and determination ofmagnetically responsive solid phase dispersal prior to transfer from thereagent well or after resuspension in the reaction well. The later maybe measured by optical or magnetic measurement of compartment contents.

External input can include operator input such as correction of amistakenly entered sample type or sample dilution factor.

Any yet unprocessed portion of a protocol may be subject to a branch.Branches may be limited to activities within a pitch or may spanactivities between pitches. Branches may alter transfers between lanesand may combine some or all of these variations. Protocols can includemultiple conditional branches.

In some embodiments, conditional branches may be limited to aborting aprotocol in progress if a fatal condition is met. For example, if thesystem detected that no millitip 220 is present in an assay cartridge,processing of that cartridge may be aborted immediately or at the nextavailable transfer window. Rather than further processing an assaycartridge 200 where no test result could be determined, the system mightuse the transfer shuttle to move that assay cartridge to the waste lanedirectly. A replacement assay cartridge could then be launched duringthe next available pitch interval to start the protocol anew.

In other embodiments, anomalies may occur that are not fatal to furtherprocessing. For example if the system failed to detect a resuspensionbuffer in a compartment of an assay cartridge 200, the system mightalter the processing protocol to provide that resuspension buffer fromanother compartment containing a reserve supply. Similarly, processingmay continue using resuspension buffer from another source such as adifferent assay cartridge 200, a reagent pack 400, or a bulk supplybottle.

In some cases, such as when reserve stocks of reagents are drawn from areagent pack 400, the system might route an assay cartridge 200 toanother processing lane 116 to provide the reserve reagent. Depending onlane availability and the tolerance of the protocol to delay, reroutingof an assay cartridge may occur either within a pitch interval or at anormal pitch interval transition. Some protocols may be tolerant ofdelay in some operations. For example, some protocols may toleratedelays after washing of solid phase but before resuspension of the solidphase. This gives an opportunity to resume processing after a delay toobtain resuspension buffer from another source. This advantageouslyavoids loss of expended reagents, sample, and time when results are notat risk.

In some embodiments, protocols may include loops. Loops are processingactivity where an assay cartridge 200 returns to a processing lane 116used during an early pitch in a later pitch. One example of a loop isthe process for routing an assay cartridge 200 from a cartridge loadinglane 116(f) to a different processing lane, then returning it to thecartridge loading lane 116(f), as described above. In another example ofa protocol that includes a loop a given assay cartridge 200 may berouted to a processing lane X at pitch N and returned to processing laneX at a pitch N+Z, where Z is a positive number. In some embodiments,protocols may include multiple returns one or more processing lanes.Loops may include conditional branches including conditional branchesthat terminate or extend loops. The protocol flexibility provided bybranching and looping beneficially allows a large variety of processing,including processing developed after the system is deployed. Thisassures that the system will keep current in its processing capabilityas new assay types are developed.

In alternative embodiments a pipeline design could advance all assaycartridges within a protocol by aligning involved lanes and displacingassay cartridges to adjacently aligned lanes. A pipeline style designmay transfer assay cartridges 200 singly or in groups. Anotheralternative could utilize multiple parallel shuttles attached to acommon transport. The common transport may displace the parallelshuttles by one or lane increments. This alternative allows selectivetransfer of individual assay cartridges between adjacent lanes, and masstransfer of each assay cartridge to its neighboring lane.

In the preferred random access design shown in FIG. 1( b), the transfershuttle 50 transfers assay cartridges 200 in a time-staggered fashion inorder to avoid conflicts. For any particular lane used in a protocol,the transfer shuttle loads successive assay cartridges at fixedintervals. The interval may be the same irrespective of the processinglanes involved. This interval, also called the pitch interval, may be ofany length, but is at least equal to the product of the time requiredfor the transfer shuttle 50 to perform a transfer operation and themaximum number of processing lanes 116 used in an extraction andpurification protocol. The time within a pitch interval may besubdivided in order to schedule the performance of multiple operationsupon an assay cartridge within a single pitch interval. For example, anassay cartridge 200 may undergo multiple fluid transfers while held in aprocessing lane 116 during a single pitch interval. As noted above, insome circumstances a pitch interval may be divided between two assaycartridges 200 using a switching operation. The use of time-staggeredtransfer with a fixed pitch interval advantageously allows a singletransfer shuttle to complete all transfers while maintaining aconsistent residence time for an assay cartridge in each processinglane. The use of a fixed pitch interval also advantageously simplifiesscheduling of multiple processes that are being performed simultaneouslywithin the system. The use of time-staggered transfer implies thatoperations on different assay cartridges in different processing lanesmay overlap in time. Some operations may proceed within one processinglane in the same time interval that the transfer shuttle 50 uses totransfer a different assay cartridge from a second processing lane to athird processing lane.

In one embodiment, the pitch interval is 150 seconds. The length of thispitch interval may be greater than the product of the time required forthe transfer shuttle 50 to perform a transfer operation and the maximumnumber of processing lanes 116 used in an extraction and purificationprotocol. In such an embodiment, the transfer shuttle may be idle atleast part of the time.

The system may reserve fixed transfer windows for each possible transfershuttle 50 operation. The preferred length of a transfer window isapproximately five seconds. If an assay cartridge 200 were present in aprocessing lane 116, the transfer shuttle 50 would transfer it to thenext processing lane in the protocol during the window associated withthat pair of processing lanes. For example, a transfer of an assaycartridge 200 from the elution lane 116(e) to the amplificationpreparation lane 116(g) may occur in a transfer window beginning 100seconds after pitch start. If, however, no assay cartridge 200 werepresent in the elution lane 116(e) during a particular pitch, thetransfer shuttle 50 would be idle during the transfer window. Dependingon the distribution of assay cartridges in the processing lanes, thetransfer shuttle may be active during each transfer window, during someof the transfer windows, or during none of the transfer windows. Thelast occurs only if no assay cartridges are in process.

The dedication of transfer windows within a pitch interval to pairs oflanes may require that the destination lane for each transfer be vacantbefore the transfer window occurs. Each processing lane 116, except thefirst and last processing lanes in a protocol, may need two transferwindows. The first transfer window allows transfer of an assay cartridge200, if one were present, out of the processing lane to a successorlane. The second transfer window allows transfer of an assay cartridge200, if one were present, into the processing lane from a predecessorlane. A consequence of this “empty before filling” requirement is thatthe system dedicates the earliest transfer window in a pitch interval tothe last processing lane pair in a protocol. This creates a “hole” inthe next to last processing lane. To account for this the system mayassign subsequent transfer windows in reverse order of the processinglane usage, so that the hole propagates through processing lanes insuccessive transfer windows until it reaches the first lane in theprotocol. The next transfer window may then occur in the following pitchinterval. In an alternative embodiment, the use of a transfer shuttle 50with multiple positions for assay cartridges 200 may allow the transfershuttle to act as temporary storage for assay cartridges beingtransferred, permitting assay cartridge switching between processinglanes as described above. Such a switching operation may take placewithin a single pitch interval.

As noted above, different protocols may route assay cartridges 200through different sequences of processing lanes. The system may transferassay cartridges among processing lanes despite a difference inprocessing lane sequence between protocols by fixing the transferwindows for transfers that are common to all protocols, by sharingtransfer windows among processing lane pairs, by delaying the start ofan instance of a protocol for one or more pitches to avoid timingconflicts, and by allocating multiple transfer windows to conflictingprocessing lane pairs.

Some transfers may be common to all protocols. For example, assaycartridge 200 disposal in the waste lane 116(c) may always followamplification mixture preparation in the amplification preparation lane116(g). Amplification mixture preparation in the amplificationpreparation lane 116(g) may, in turn, always follow nucleic acid elutionin the elution lane 116(e), which may always follow a small magnet washin the wash lane 116(b). Transfers among these lanes need not presentany special timing problems; the system may use fixed transfer windowsfor such transfers. The system may also use fixed transfer windows whentransferring assay cartridges among lanes used only by a singleprotocol. Transfers among these lanes present no timing conflicts.

The system may share a fixed transfer window when a common source lanetransfers to two or more different destination lanes. This need notpresent a timing conflict, as the system may transfer an assay cartridge200 in the source lane to only one of these destination lanes at a givenpoint in the protocol. The source lane can maintain a single transferwindow to unload; the destination lanes may share this single fixedtransfer window to receive an assay cartridge from the source lane.

The system may also share a fixed transfer window when a commondestination lane receives transfers from more than one source lane. Thiscan generate a timing conflict. In one embodiment, the destination lanemaintains a fixed transfer window to avoid shifts in timing that mightpropagate to subsequent transfers and create further conflicts. Sincethe destination lane may receive only one transfer, the system mayschedule protocol instances so that only one of the source lanescontains an assay cartridge. This may require that the system look aheadto determine a possible conflict and delay the start of an instance of aprotocol for one or more pitch intervals to avoid the conflict.

The system may allocate multiple transfer windows when a protocolinserts the use of one or more non-common processing lanes between lanesthat are common to another protocol. These inserted lanes require atleast one pitch interval, but the subsequent return to the common lanesrequires preservation of the common lane transfer windows in order tominimize timing conflicts. Providing more than one transfer windowallows the system to select among transfer windows to minimizeconflicts. The system may shift the transfer from the last common lanebefore the insert to the later transfer window. The system may return tothe common lane timing when the assay cartridge returns to the commonlanes. For example, the RNA protocol may insert a non-common step bytransferring the assay cartridge 200 sequentially through processinglanes 8, 9, and 10. DNA protocols may not use lane 9, but rather movethe assay cartridge 200 directly from lane 8 to lane 10. In thisinstance the system may include two transfer windows to move assaycartridges out of lane 8. The first window begins at 110 seconds afterpitch start. The second transfer window begins at 115 seconds afterpitch start. The RNA protocol uses the later transfer window to move theassay cartridge from lane 8 to lane 9 at 115 seconds after pitch start.The DNA protocols use the earlier transfer window. Every protocoltransfers an assay cartridge into lane 10 at the transfer windowbeginning 110 seconds after pitch start. The multiple transfer windowsfor lane 8 produce a dead period in the lane 8 pitch interval for theDNA protocols. During this dead period, lane 8 sits empty. The deadperiod does not upset processing timing because it is consistent foreach instance of the DNA protocols.

As discussed above, a switch between protocols may cause a timingconflict that the system may resolve by delaying a protocol start forone or more pitch intervals. Such a delay may reduce system throughput.The system minimizes the number of such delays by scheduling assays soas to minimize any delays. In some embodiments, the system starts allpending assays that use the same protocol before starting any pendingassays that use a different protocol.

Within a pitch interval, and subject only to the timing of transferwindows, a protocol may use a processing lane to perform any operationsof which the lane is capable. These operations may be in any sequenceand may be of any duration. The system may perform two or moreconsecutive sets of processing steps in a single processing lane overmultiple pitch intervals without transferring the assay cartridge 200.The system thus provides two levels of protocol flexibility: first, aprotocol may selectively route assay cartridges among processing lanes;and second, a protocol may freely select operations within a processinglane. First and second assay cartridges may be used to process samplesaccording to first and second protocols, wherein the first and secondprotocols may be different.

As noted above, while the system may transfer an assay cartridge 200between any two processing lanes 116 in order to accommodate a varietyof sample types and assay chemistries, the general workflow of theisolation process may be similar. This provides that certain generalsteps may occur in the same sequence. Nucleic acid extraction andisolation methods are known and described, e.g., in Merel et al. (1996)Clinical Chemistry 42:1285-6; Ausubel et al. Current Protocols inMolecular Biology (2003 ed.); Sambrook et al. Molecular Cloning (3^(rd)ed.); Bailey et al. (2003) J. Assoc. Lab. Automation 8:113-20. Theprocess generally includes steps of sample treatment, binding of thenucleic acids in the sample to a solid or suspended particulate phase,separation of the bound nucleic acids from unbound components of thesample, washing the solid or suspended particulate phase, and elution orrelease of the nucleic acid back into solution. The purpose of thesesteps is to release nucleic acids from cells, nuclei, or sample matrix,to reduce or eliminate components that may interfere with nucleic acidamplification or detection, and to adjust the concentration of nucleicacids relative to the concentration in the original sample. Variationsof the described process and other nucleic acid isolation protocols arealso within the scope of the invention. Variations may include changesin the volumes of materials transferred, in the conditions of chemicalprocessing steps, in the sequence of operations, in the number of washsteps, and other changes.

In one embodiment, the system extracts and purifies nucleic acids bymixing magnetically responsive microparticles with an aliquot of sampleand reagents under environmental conditions that favor binding ofnucleic acids to the solid phase. When extraction and purification areperformed in a cartridge such as the one shown in FIG. 4( a)-1, reagentstransferred from the reagent wells 204, 208, 209 to the reaction well202 of the assay cartridge 200 in early steps of the protocol mayprovide conditions that favor binding of the target nucleic acidsequence to the magnetically responsive microparticles. Reagents may bearranged in the wells of the assay cartridge 200 in an order thatreflects their use, so that droplets that accidentally fall duringreagent delivery operations only land in previously emptied wells.

Once the nucleic acids bind to the solid phase the system may transferthe cartridge to wash lanes, such as 116(a) and 116(b) of FIG. 1( b), toremove unbound material by applying a magnetic field to the reactionmixture; magnetic microparticles respond to the applied magnetic fieldby moving within the reaction mixture, thereby segregating the solidphase from the bulk liquid. The system can then remove the bulk liquidby aspiration, leaving behind the solid phase. An embodiment of aprocessing lane that includes such a magnetic separator is shown in FIG.10( b) and described in more detail above. In subsequent steps, thesystem may wash the solid phase by adding a wash liquid, re-suspendingsolid phase to form a suspension in the wash liquid, and againsegregating the solid phase followed by aspiration of the liquid portionof the reaction mixture while leaving behind the solid phase. This washstep may be repeated several times, and may involve the use of one orwash liquids. In some embodiments, expended wash liquids are returned topreviously emptied wells of the assay cartridge 200 for eventualdisposal. When washing (block 1820) is complete, the system may transferthe cartridge to an elution lane 116(e) and add an eluent, whichreleases the nucleic acid from the solid phase and back into solutionwithin the eluent volume (block 1822). The system may complete thenucleic acid extraction and purification process by transferring thecartridge to an amplification preparation lane 116(g) and againsegregating the solid phase through application of a magnetic field,followed by aspiration of the eluent volume and transfer of the eluentvolume containing the isolated nucleic acid to a reaction vessel forfurther processing (block 1824). In an alternative embodiment, thesystem may transfer reagents required for amplification to a reactionvessel prior to transfer of the eluent volume containing the isolatednucleic acid to the reaction vessel.

The solid phase can be a magnetically responsive solid phase. Underthese circumstances, an applied magnetic field can act as a controllableswitch to selectively anchor a magnetically responsive solid phase. Ifthe solid phase is a suspension of magnetically responsivemicroparticles these may form a distinctive “pellet” against a desiredlocation on the interior wall of a container on application of amagnetic field. The location, shape, and size of this pellet can becontrolled by controlling the distribution and intensity of the magneticfield, advantageously permitting the system to generate pellets of solidphase at different locations within a container, and with desirablecharacteristics for avoiding nonspecific aggregation of the particlesand for resuspension on removal of the magnetic field. Thisadvantageously simplifies automation because the system may simply applya magnetic field either by disposing the magnetically responsive solidphase in proximity to magnetic materials or by activating anelectromagnet.

Although a magnetically responsive solid phase is preferred, other solidphases may also be suitable. For example, the system may manipulate thesolid phase by settling under gravity or centrifugation, by filtration,by size exclusion chromatography, by optical tweezers, byelectrophoresis, by dielectrophoresis, by flow cytometry based sorting,by mechanical obstruction such as the use of solid phases too large tofit within a pipette during separation, or by any of a number of othermethods.

The magnetically responsive solid phase is preferably a suspension ofmagnetically responsive microparticles. They advantageously simplifyautomation as the system may transfer a measured amount of solid phaseby simple pipetting, which is a well-established and repeatable process.Pipetting has the further benefit of commonality with other liquidreagent transfers. That is, the system needs no additional devices totransfer the solid phase. A suspension of magnetically responsivemicroparticles has the further advantage of improving assay speed andprecision by providing a more uniform interaction between solid phaseand solvated components of the liquid reaction mixture. A dispersedsuspension of microparticles reduces the time required for nucleic acidisolation by minimizing diffusion distances between reactants. Thisdispersion also improves uniformity by providing each element of theliquid reaction mixture with approximately equal access to the solidphase as each other liquid element. This improved reaction uniformitydirectly enhances assay reproducibility, and hence precision.Magnetically responsive microparticles are known in the art and arecommercially available. Microparticles for nucleic acid binding can befunctionalized with various species that will attract and bind nucleicacids, including, but not limited to, nucleic acid sequences, proteins,dyes, hydrophilic groups, hydrophobic groups, and charged groups.

Processing a sample in this fashion provides the opportunity toconcentrate the isolated target nucleic acid in a reduced volume. Thesystem may adjust nucleic acid concentration by isolating nucleic acidsfrom relatively large sample volumes and eluting the isolated nucleicacids from the solid or suspended particulate phase in a relativelysmall volume. This has beneficial effects of reducing assay time,increasing assay sensitivity, and improving assay precision. In someembodiments, the volume of sample initially transferred is about 1 mLand the volume of eluent added is about 404. In some embodiments, thevolume of eluent transferred to the amplification vessel is smaller thanthe volume of eluent added, in order to account for dead volume in thereaction vessel and minimize the chances of inadvertent transfer ofsolid phase to the reaction vessel. In some embodiments, the volume ofeluent transferred is about 254.

Adjusting nucleic acid concentration can advantageously reduce assaytime by reducing the volume of subsequent reactions. PCR is dependent oncycling the reaction volume through a series of temperature changes.Small amplification reaction volumes permit reduced thermal pathlengths,leading to more rapid thermal equilibration of the entire reactionvolume and hence reduced temperature cycle time. Higher concentrationsof target nucleic acids within the amplification reaction volume canalso reduce the number of amplification cycles required for detection,as the growth curve that characterizes successful PCR amplification willbecome evident earlier in the process.

As discussed above, a short thermal pathlength allows rapid thermalequilibration of a reaction volume. This in turn enables rapidtemperature changes during amplification reactions. Thermalcycling-based amplification methods typically cycle amplificationreaction mixtures through a number of target temperatures, each targettemperature supporting one or more phases of the amplification reaction.A typical PCR amplification may require 50 or more of these temperaturecycles. Rapid temperature changes reduce the time required for eachcycle of amplification. This reduced cycle time is especially desirableas even small time savings accumulate rapidly over multipleamplification cycles, thus reducing the overall time required to produceanswers.

Adjusting nucleic acid concentration can increase assay sensitivity bykeeping the number of amplification cycles within a reproducible range.Exponential nucleic acid amplification, such as PCR, is subject to noiseand to nonspecific amplification that may produce an erroneous signal ifthe reaction is allowed to continue for a large number of cycles, evenin the absence of the target nucleic acid. As a result, attempting toimprove the sensitivity of a PCR-based assay by simply extending thenumber of amplification cycles soon encounters a limiting condition. Byincluding a higher concentration of target sequences in the initialamplification mixture, a signal that is attributable to targetamplification can appear in earlier cycles, thus avoiding erroneousresults from spurious amplification events. The higher target sequenceconcentration attainable by adjusting the nucleic acid concentrationincreases confidence that signals observed reflect the actual presenceof target sequences rather than spurious events. Since assay sensitivitydepends, at least in part, on distinguishing target-based specificsignal from non-target spurious signals, higher initial target sequenceconcentrations improve overall assay sensitivity.

Adjusting nucleic acid concentration also improves assay precision byreducing the effect of sampling error. Amplification based assays permitthe detection of extremely low concentrations of target sequence. Sometarget nucleic acid sequences may be present at such low concentrationsthat individual aliquots taken from the same sample may have significantvariations in the number of target sequences present. This variationestablishes an irreducible minimum of imprecision in determination ofthe target concentration in the aliquot. For example, where eachmilliliter of sample contains 1000 copies of a target nucleic acidsequence, 54 aliquots of such a sample would contain, on average, fivecopies. Basic statistics show, however, that less than 18% of individual54 aliquots would contain this average number of copies. About 3% of 5μL aliquots would contain at least ten copies; tests on these aliquotswould overestimate target sequence concentration by a factor of two ormore. A small fraction of 5 μL aliquots would contain no target nucleicacid sequences at all, so that mere detection of the presence of thesequence would be impossible. One way to reduce the effect of samplingerror is to increase the volume of the sample aliquot. However, thiswould necessarily increase the final reaction volume. For the reasonsnoted above, this is undesirable. Adjusting nucleic acid concentrationallows use of a large initial source sample aliquot, the nucleic acidsof which are released by sample processing into a smaller test aliquotto increase the number of target nucleic acid sequence copies in theamplification mixture, while retaining the time savings and otherbenefits of small amplification volumes.

As noted above, the system may accomplish the goal of adjusting nucleicacid concentration by isolating nucleic acids using a solid phase. Thissolid phase may be a particulate or microparticulate phase that canremain in fluid suspension for a time, which advantageously simplifieshandling and improves reaction kinetics. Solid phase processing permitsseparation and exchange of liquid components of a reaction mixture whileretaining specific reactants, such as nucleic acids, that are bound tothe solid phase. This binding may be physical or chemical, but theseparation process is mechanical. Solid phase processing is beneficialbecause its mechanical separation process is readily automatable, andcan provide a cleaner separation than the precipitation or liquid/liquidphase separations of conventional chemical processes.

Although solid phase processing is preferred, other methods of adjustingnucleic acid concentration may also be suitable. For example, the systemmay precipitate nucleic acids and separate the precipitate from theremaining supernatant by filtration or centrifugation. Alternatively,the system may extract nucleic acids by differential solubility inorganic and aqueous phases or by separating the nucleic acids from otherconstituents by electrophoresis, column chromatography, or by any of anumber of other methods. In order to utilize this method to concentrateisolated nucleic acids, the system can have the capacity to accuratelydispense both large and small volumes.

Accordingly, the system may include both large volume pipettors thatutilize millitips 220 provided in the assay cartridge 200 (as shown inFIG. 4( a)-1) and small volume pipettors that utilize microtips 542 thatare incorporated into the processing lanes 116 or have access to them.Microtips 542 may be supplied in microtip racks 550, as shown in FIG.13( f), that are loaded onto the system by the user 1840. In anembodiment shown in FIG. 1( c) the system includes a microtip storageunit 120 for this purpose. A detailed description of a preferredembodiment of a microtip storage unit is found above and in FIGS. 13(a), 13(b), and 13(c). The system may automatically deposits expendedmicrotips 542 into solid waste, such as the solid waste container 92shown in FIG. 1( d), but users may need to unload empty microtip racks550. Alternatively, the system may dispose of used mictrotips within thewells of an assay cartridge 200. The multiple slots within the microtipstorage unit 120 allow the system to use all microtips 542 within amicrotip rack 550 without concern of running out of microtips 542;microtip racks 550 in other slots provide a reserve capacity.

Users may unload empty microtip racks 550 once the system has used allmicrotips 542 in a microtip rack 550. In operation, a user may requestthat the instrument provide a loading opportunity. In preparing for aloading opportunity, the system may release empty microtip racks 550from the microtip storage unit 20 by releasing the rack clasp 554associated with the selected microtip racks 550. During a loadingopportunity, a user may open the access cover 556 and view indicatorlamps associated with each loaded microtip rack 550. The user may removeany released microtip rack 550 and insert any new microtip racks 550. Insome embodiments, users may not reload microtip racks previouslyunloaded back onto the system. This advantageously limits thepossibility of contamination from user handling of exposed microtips.

Following isolation of the target nucleic acid, at least a portion ofthe elution volume containing the target nucleic acid is transferred toa reaction vessel 221 that may be provided on the assay cartridge 200,as shown in FIG. 4( a)-1. In some embodiments this takes place in anamplification preparation lane, such as 116(g) of FIG. 1( b), which mayalso be accessible to the XYZ transport device 40. Other materialsuseful for the amplification reaction may also be added to the reactionvessel 221. In some embodiments, these amplification materials aretransferred to the reaction vessel 221 prior to the transfer of theelution volume to the reaction vessel 221. Such materials may include,but are not limited to polymerases required for nucleic acidreplication, target-specific primer sequences, target-specific probesequences, nucleotide triphosphates, and other materials that supportthe amplification reaction. These materials may be stored in the reagentstorage module 10 and transferred using the XYZ transport device 40.Following the addition of processed sample and all necessary reagentsthe reaction vessel 221 may be closed using a plug 222. This plug 222can be provided on the assay cartridge 200, and may include a handlingfeature 222(f) that allows it to be manipulated by the XYZ transportdevice 40. Insertion of the plug 222 into the reaction vessel 221 mayseal the reaction vessel for the remainder of its time on the system.

After sealing, the reaction vessel 221 proceeds to the amplification anddetection portion of the system (block 1832). Amplification phaseprocessing centers on the reaction vessel 221 and the thermal cyclers.Processing in the amplification phase may be mechanically simplecompared to the isolation phase. Once the amplification preparation lane116(g) mixes the isolated nucleic acid with amplification reagents inthe reaction vessel, the system may seal the reaction vessel 221 andtransport it to an available thermal cycler module. In a preferredembodiment, the system has multiple thermal cycler modules, which may bearranged in a garage 1200 as shown in FIG. 16( c). The performance ofthese thermal cycler modules 1300 may be matched, so that the path ofthe reaction vessel after leaving the processing lanes 116 may lead toany one of the thermal cycler modules 1300. The system may then lock thevessel into the thermal cycler module 1300 and begin the process ofthermal cycling and monitoring (block 1832). The thermal cycling andmonitoring continues until the earlier of signal detection or a pre-setnumber of thermal cycles without signal detection.

In some embodiments, particularly those associated with reversetranscription of isolate RNA sequences, the thermal cycler may heat theamplification vessel to a fixed temperature prior to initiatingamplification by, for example, thermal cycling.

In some embodiments, the system monitors the progress of theamplification by illuminating the reaction vessel 221 with excitationlight at selected points within each thermal cycle. The instrument mayselect these points based on the part of the thermal cycle and on themeasured temperature in the amplification vessel. In some embodiments,the system measures the signal during the same portion of each thermalcycle, but the timing within the portion may vary so that theamplification vessel has a measured temperature equal to a preselectedtemperature at the time of measurement. This has the benefit of reducingvariations in measurement that might otherwise contribute to assayimprecision. In another embodiment, the system measures the signalwithin a defined portion of a defined temperature versus time profilethat the thermal cycler is directed to follow. This has the benefit ofproviding consistent thermal cycling times, thereby simplifyingscheduling. The system may combine measurements from multiple thermalcycles to assign one or more values to the measured reaction (block1834). Numerous methods of combining measurements are known in the art.

After removal of the sealed reaction vessel 221 the expended assaycartridge may be transferred to waste. In one embodiment, shown in FIG.1( b), the transfer shuttle 50 moves the expended cartridge 1826 to awaste lane 116(c). As noted above, the waste lane 116(c) may beconfigured so that once an assay cartridge 200 is placed within it theassay cartridge 200 cannot be returned to the transfer shuttle 50. Anembodiment of such a waste lane is shown in FIGS. 14( a), 14(b), and14(c). The waste lane may be supplied with an aspiration probe 986 toremove remaining fluid contents of the cartridge to liquid waste 1830.The emptied assay cartridge 200 may then be discarded (block 1848) tothe solid waste container 882. In some embodiments, the expended assaycartridge 200 is simply transferred to the solid waste container 882along with any residual liquids it may contain.

After completion of thermal cycling, the system may release the reactionvessel 221 from the thermal cycler, and the XYZ transport device 40 maytransfer (block 1850) the expended reaction vessel 221 to the solidwaste container 882, thereby ending the processing of a specific sample.In some embodiments, the expended reaction vessel is disposed of bytransferring it to a dedicated wasted container, which may be designedto avoid damage to the expended reaction vessel. In other embodiments,the expended reaction vessel is removed from the system by transferringit to an unloading rack, where it may be retrieved by the user forfurther analysis.

EXAMPLES

Each of the examples below summarizes the processing steps in aprotocol. The processing steps include extraction and isolation ofnucleic acids, set up of the amplification mixture, transfer of theamplification mixture to a thermal cycler, amplification and detection,and waste disposal.

Example 1 Gram Positive DNA: Group B Streptococcus Assay

Pitch Lane/Device Operations 1 CLU Presentation Transfer sample aliquotto assay Lane cartridge reaction well Transfer process controls fromreagent pack to assay cartridge reaction well (XYZ gantry) Transferenzyme from reagent pack to assay cartridge reaction well (XYZ gantry)Mix contents of assay cartridge reaction well Transfer assay cartridgeto shuttle 2 70° C. Processing Retrieve assay cartridge from shuttleLane Temperature stabilize at 70° C. (90 seconds) Mix paramagneticparticles in assay cartridge reagent well Transfer buffer andparamagnetic particles from assay cartridge reagent wells to assaycartridge reaction well Transfer assay cartridge to shuttle 3 Wash Lane1 Retrieve assay cartridge from shuttle (Large Magnet) Mix contents ofassay cartridge reaction well Apply magnet Aspirate liquid from assaycartridge reaction well Transfer assay cartridge to shuttle 4 Wash Lane2 Retrieve assay cartridge from shuttle (Large Magnet) Transfer washbuffer 1 from assay cartridge reagent well to assay cartridge reactionwell Mix contents of assay cartridge reaction well Apply magnet Aspirateliquid from assay cartridge reaction well Transfer assay cartridge toshuttle 5 Wash Lane 3 Retrieve assay cartridge from shuttle (LargeMagnet) Transfer wash buffer 2 from assay cartridge reagent well toassay cartridge reaction well Mix contents of assay cartridge reactionwell Apply magnet Aspirate liquid from assay cartridge reaction wellTransfer assay cartridge to shuttle 6 Wash Lane 4 Retrieve assaycartridge from shuttle (Small Magnet) Transfer wash buffer 3 from assaycartridge reagent well to assay cartridge reaction well Mix contents ofassay cartridge reaction well Apply magnet Aspirate liquid from assaycartridge reaction well Transfer assay cartridge to shuttle 7 ElutionLane Retrieve assay cartridge from shuttle (Large Magnet) Transferelution buffer from assay cartridge reagent well to assay cartridgereaction well Mix contents of assay cartridge reaction well Apply magnetTransfer liquid from assay cartridge reaction well to reaction vesselTransfer assay cartridge to shuttle 8 PCR Preparation Retrieve assaycartridge from shuttle Lane Transfer PCR reagents from reagent pack toreaction vessel (XYZ gantry) Transfer plug to reaction vessel and sealTransfer sealed reaction vessel to thermal cycler (XYZ Gantry) Transferassay cartridge to shuttle 9 Waste Lane Retrieve assay cartridge fromshuttle Transfer assay cartridge to waste 9 to N Thermal Cycler Amplifyand monitor contents of reaction vesselTo incorporate the use of the Cartridge Warming Lane, the processing ofa series of assay cartridges is interleaved. Within a given pitch (X),at about 50 seconds after moving into the CLU presentation lane andreceiving a sample aliquot, the assay cartridge (N) is moved to one ofthe two positions of the transfer shuttle. The shuttle moves to theCartridge Warming Lane and retrieves the previous assay cartridge (N−1)in the series from the cartridge heater into the remaining openposition, then transfers the current assay cartridge (N) to thecartridge heater. The previous assay cartridge (N−1) is then returned tothe CLU presentation lane by the 60 second mark of the pitch (X) forfurther processing through the end of pitch (X), after which it moves onto the next lane in the protocol designated for assay cartridge (N−1) atthe start of pitch (X+1). This leaves the transfer shuttle empty. Athird assay cartridge (N+1) is moved to the CLU presentation lane at thestart of pitch (X+1), receives a sample aliquot, and is moved to thetransfer shuttle at about 50 seconds after the start of the pitch (X+1).The assay cartridge (N) is returned to the CLU presentation lane at the60 second mark of the subsequent pitch (X+1) for further processingafter it is switched in the Cartridge Warming Lane for the next assaycartridge (N+1) in the series, and so on.

Example 2 DNA: CT-NG Assay

Pitch Lane/Device Operations 1 CLU Presentation Transfer sample aliquotto assay Lane cartridge reaction well Transfer process controls fromreagent pack to assay cartridge reaction well (XYZ gantry) Transferdilution buffer from assay cartridge reagent well to assay cartridgereaction well Transfer digestion buffer from assay cartridge reagentwell to assay cartridge reaction well Transfer enzyme from reagent packto assay cartridge reaction well Mix contents of assay cartridgereaction well Transfer assay cartridge to shuttle 2 37° C. ProcessingRetrieve assay cartridge from shuttle Lane Mix binding buffer andparamagnetic particles in assay cartridge reagent wells Transfer bindingbuffer and paramagnetic particles from assay cartridge reagent wells toassay cartridge reaction well Mix contents of assay cartridge reactionwell Transfer assay cartridge to shuttle 3 Wash Lane 1 Retrieve assaycartridge from shuttle (Large Magnet) Apply magnet Aspirate liquid fromassay cartridge reaction well Transfer assay cartridge to shuttle 4 WashLane 2 Retrieve assay cartridge from shuttle (Large Magnet) Mix washbuffer 1 in assay cartridge reagent well Transfer wash buffer 1 fromassay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 5Wash Lane 3 Retrieve assay cartridge from shuttle (Large Magnet) Mixwash buffer 2 in assay cartridge reagent well Transfer wash buffer 2from assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 6Wash Lane 4 Retrieve assay cartridge from shuttle (Small Magnet) Mixwash buffer 3 in assay cartridge reagent well Transfer wash buffer 3from assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 7Elution Lane Retrieve assay cartridge from shuttle (Large Magnet) Mixelution buffer in assay cartridge reagent well Transfer elution bufferfrom assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Transfer liquidfrom assay cartridge reaction well to reaction vessel Transfer assaycartridge to shuttle 8 PCR Preparation Retrieve assay cartridge fromshuttle Lane Transfer PCR reagents from reagent pack to reaction vessel(XYZ gantry) Transfer plug to reaction vessel and seal Transfer sealedreaction vessel to thermal cycler (XYZ Gantry) Transfer assay cartridgeto shuttle 9 Waste Lane Retrieve assay cartridge from shuttle Transferassay cartridge to waste 9 to N Thermal Cycler Amplify and monitorcontents of reaction vessel N + 1 XYZ Gantry Transfer reaction vessel towasteTo incorporate the use of the Cartridge Warming Lane, the processing ofa series of assay cartridges is interleaved. Within a given pitch (X),at about 50 seconds after moving into the CLU presentation lane andreceiving a sample aliquot, the assay cartridge (N) is moved to one ofthe two positions of the transfer shuttle. The shuttle moves to theCartridge Warming Lane and retrieves the previous assay cartridge (N−1)in the series from the cartridge heater into the remaining openposition, then transfers the current assay cartridge (N) to thecartridge heater. The previous assay cartridge (N−1) is then returned tothe CLU presentation lane by the 60 second mark of the pitch (X) forfurther processing through the end of pitch (X), after which it moves onto the next lane in the protocol designated for assay cartridge (N−1) atthe start of pitch (X+1). This leaves the transfer shuttle empty. Athird assay cartridge (N+1) is moved to the CLU presentation lane at thestart of pitch (X+1), receives a sample aliquot, and is moved to thetransfer shuttle at about 50 seconds after the start of the pitch (X+1).The assay cartridge (N) is returned to the CLU presentation lane at the60 second mark of the subsequent pitch (X+1) for further processingafter it is switched in the Cartridge Warming Lane for the next assaycartridge (N+1) in the series, and so on.

Example 3 RNA: Hepatitis C Virus Assay

Pitch Lane/Device Operations 1 CLU Presentation Transfer sample aliquotto assay Lane cartridge reaction well Transfer process controls fromreagent pack to assay cartridge reaction well (XYZ gantry) Transferdilution buffer from assay cartridge reagent well to assay cartridgereaction well Transfer digestion buffer from assay cartridge reagentwell to assay cartridge reaction well Transfer enzyme from reagent packto assay cartridge reaction well Mix contents of assay cartridgereaction well Transfer assay cartridge to shuttle 2 70° C. ProcessingRetrieve assay cartridge from shuttle Lane Mix binding buffer andparamagnetic particles in assay cartridge reagent well Transfer bindingbuffer and paramagnetic particles from assay cartridge reagent wells toassay cartridge reaction well Mix contents of assay cartridge reactionwell Transfer assay cartridge to shuttle 3 Wash Lane 1 Retrieve assaycartridge from shuttle (Large Magnet) Apply magnet Aspirate liquid fromassay cartridge reaction well Transfer assay cartridge to shuttle 4 WashLane 2 Retrieve assay cartridge from shuttle (Large Magnet) Mix washbuffer 1 in assay cartridge reagent well Transfer wash buffer 1 fromassay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 5Wash Lane 3 Retrieve assay cartridge from shuttle (Large Magnet) Mixwash buffer 2 in assay cartridge reagent well Transfer wash buffer 2from assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 6Wash Lane 4 Retrieve assay cartridge from shuttle (Small Magnet) Mixwash buffer 3 in assay cartridge reagent well Transfer wash buffer 3from assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Aspirate liquidfrom assay cartridge reaction well Transfer assay cartridge to shuttle 7Elution Lane Retrieve assay cartridge from shuttle (Large Magnet) Mixelution buffer in assay cartridge reagent well Transfer elution bufferfrom assay cartridge reagent well to assay cartridge reaction well Mixcontents of assay cartridge reaction well Apply magnet Transfer liquidfrom assay cartridge reaction well to reaction vessel Transfer assaycartridge to shuttle 8 PCR Preparation Retrieve assay cartridge fromshuttle Lane Transfer PCR reagents from reagent pack to reaction vessel(XYZ gantry) Transfer plug to reaction vessel and seal Transfer sealedreaction vessel to thermal cycler (XYZ Gantry) Transfer assay cartridgeto shuttle 9 Waste Lane Retrieve assay cartridge from shuttle Transferassay cartridge to waste 9 to N Thermal Cycler Fixed temperature forreverse transcription Amplify and monitor contents of reaction vesselN + 1 XYZ Gantry Transfer reaction vessel to wasteTo incorporate the use of the Cartridge Warming Lane, the processing ofa series of assay cartridges is interleaved. Within a given pitch (X),at about 50 seconds after moving into the CLU presentation lane andreceiving a sample aliquot, the assay cartridge (N) is moved to one ofthe two positions of the transfer shuttle. The shuttle moves to theCartridge Warming Lane and retrieves the previous assay cartridge (N−1)in the series from the cartridge heater into the remaining openposition, then transfers the current assay cartridge (N) to thecartridge heater. The previous assay cartridge (N−1) is then returned tothe CLU presentation lane by the 60 second mark of the pitch (X) forfurther processing through the end of pitch (X), after which it moves onto the next lane in the protocol designated for assay cartridge (N−1) atthe start of pitch (X+1). This leaves the transfer shuttle empty. Athird assay cartridge (N+1) is moved to the CLU presentation lane at thestart of pitch (X+1), receives a sample aliquot, and is moved to thetransfer shuttle at about 50 seconds after the start of the pitch (X+1).The assay cartridge (N) is returned to the CLU presentation lane at the60 second mark of the subsequent pitch (X+1) for further processingafter it is switched in the Cartridge Warming Lane for the next assaycartridge (N+1) in the series, and so on.

U. System Control Architecture

Control and coordination of the activities of the subsystems describedabove is provided by one or more computers. In one embodiment of theinvention, control of the system is distributed between a primarycontroller and a plurality of secondary controllers. The primarycontroller may include one or more computers, which provide a userinterface and transmit primary commands to secondary controllers. Eachsubsystem may incorporate a secondary controller that receives commandsfrom the primary controller. Examples of secondary controllers includecompact motion control cards, also known as a cMCCs, and cMCC-derivedcontrol cards. A secondary controller is configured to receive a primarycommand from a system computer, and then processes the primary commandto generate a series of secondary commands that are transmitted toeffectors incorporated into the subsystem in order to achieve theprimary command. Examples of primary commands received from the primarycontroller include, but are not limited to, designation of a position ofa system component or temperature of a system component. Examples ofsecondary commands generated by a secondary controller include, but arenot limited to, speed of rotation in a specific motor, duration ofrotation in a specific motor, and voltage applied to a temperaturecontrolling element. Examples of effectors acted upon by the secondarycontroller include rotary stepper motors, linear stepper motors,resistive heating elements, and thermoelectric cooling elements. Inaddition, a secondary controller may monitor feedback from thesubsystem, and utilize that feedback to generate corrective secondarycommands as necessary. Examples of feedback provided to a secondarycontroller include, but are not limited to, information related toactual position of a subsystem component or to actual temperature of asubsystem component. Secondary controllers may also be used to performanalog to digital data conversion.

Tasks such as continuous generation of secondary commands, subsequentmonitoring and correction of operations, and analog to digital dataconversion are tasks that require real time, high frequency processing.This system architecture advantageously permits the use of secondarycontrollers with specialized microprocessors, for example cMCCs andcMCC-derived control cards that are optimized for repetitive, highfrequency tasks. Secondary controllers can also utilize system on achip, or SOC, cards that combine control and analog data conversionfunctions. Control cards used in secondary controller may incorporate anonboard bus that permits expansion of the functions of the secondarycontroller. Such an expansion of function could include additionalinputs and/or outputs to and from the control card, respectively.Another example of expanded function is to provide communication with anadditional, tertiary control card. The use of a primary controller withconnections to secondary controllers advantageously permits accurate andrapid control of subsystem functions while allowing the use of a generalpurpose computer as a primary controller to provide functions such asdata storage and a familiar interface for the user.

As noted above, secondary controllers may receive data related to theperformance of their associated subsystems. This data may serve asfeedback, used to generate corrective secondary commands. Data receivedby the secondary controllers may also be transferred to the primarycontroller. This data can include data from position encoders, homingsensors, automated alignment procedures, current supplied to heatingelements, temperatures achieved by heating elements, temperatureprofiles from thermal cyclers, and number of duty cycles for specificcomponents. Such data can be used to determine if a subsystem orsubsystem component shows evidence of deteriorating performance. If sucha determination is made the system may notify the user in advance of thefailure of a subsystem or subsystem component, permitting the user toperform maintenance or schedule service on the system prior toexperiencing an actual system malfunction. This advantageously reducessystem downtime.

In some embodiments secondary controllers incorporate safety features,including shutdown commands for motors, solenoids, or heaters. A primarycontroller may cascade a global shutdown command throughout thesecondary controllers of the system. Alternatively, a global shutdowncommand may originate with or be communicated between secondarycontrollers.

In some subassemblies, the secondary controller may be associated with asensing circuit that provides feedback to the system. As describedabove, the sensing circuit can provide a signal that indicates when aportion of the subassembly contacts or approaches a liquid or a surface.In some embodiments this sensing circuit is a capacitance-based liquidsensing circuit as described above, which may include a reactive elementthat forms part of a tuned circuit in a voltage-controlled oscillator.In some embodiments, the reactive element is a liquid handling probethat forms part of the liquid sensing circuit. Alternatively, thereactive element may be a conductive extension of the subassembly thatis discarded after use. Examples of disposable conductive extensionsinclude, but are not limited to, millitips and microtips.

A sensing circuit may also be used to detect contact with or proximityto conductive surfaces. In one embodiment, the sensing circuit can beused to detect the successful attachment of conductive items to apipette mandrel that forms part of the circuit. In such an embodiment,the sensing circuit can provide a signal that indicates the successfulattachment, and subsequent release, of a conductive millitip (220 ofFIG. 6), microtip (490 of FIG. 12( b)), or reaction vessel plug (222 ofFIG. 5) to the pipette mandrel.

In another embodiment, the sensing circuit can be used to detect theapproach of a pipette mandrel which forms part of the circuit to one ormore conductive targets that are placed within the path of the pipettor.This approach can be a patterned series of movements that comprise asearch for a conductive target that is initiated once the pipettemandrel is brought into proximity to the conductive target. Suchinformation, when combined with information regarding the position of anassociated stepper motor, can be used for automating alignment of thepipettor within the system. The conductive targets may be fortuitouslylocated system components or conductive targets incorporated into thesystem for this purpose. Conductive targets can include projections thatextend from a system component. Examples of projecting conductivetargets include substantially planar tabs and cylindrical pins.Alternatively, a conductive target can be a hole or gap in an otherwisecontinuous conductive surface

The primary controller may be connected to a secondary controller by anetwork connection. This connection may convey information or mayprovide both information and power to the secondary controller. In oneembodiment, the connection is provided by a Controller Area Network bus,also known as a CAN bus, a digital serial bus that is commonly used inindustrial environments. Alternatively, the network connection betweenthe system primary controller and a secondary controller can be aUniversal Serial Bus, RS-485, Ethernet, or HSSI connection. Such networkconnections may also be used to provide communication between secondarycontrollers. Wireless connections, such as Zigbee, Firewire, orBluetooth may also be used to provide communication between a primarycontroller and a secondary controller, or between secondary controllers.Such communication between secondary controllers facilitatessynchronization of tasks throughout the system.

In one embodiment, most of the subsystems of the system can incorporatea secondary controller. Subsystems that incorporate a secondarycontroller may include, as shown in FIG. 1( b), individual processinglanes of the sample processing lane assembly 116, the cartridge transfershuttle 50, the cartridge loading unit 112, the sample presentation unit110, the XYZ transport device 40, the sample pipettor assembly 70, thereagent storage module 10, and the thermal cycler garage 30. In someembodiments, the secondary controller directs the activities of thesubassembly into which it is incorporated. Alternatively, a secondarycontroller may direct the activities of the assembly with which it isincorporated and one or more other subassemblies. For example, asecondary controller incorporated into the thermal cycler garage 30 maycontrol activity within the thermal cycler subassembly and additionallycontrol activities within the optical subsystem (FIG. 18( c)). In someembodiments a subassembly may incorporate more than one secondarycontroller, each of which directs the activities of different portionsof the subassembly. For example, the thermal cycler garage (30 of FIG.1( b)) may incorporate two secondary controllers, each responsible forthe control of a portion of the plurality of thermal cyclers housedwithin the thermal cycler garage. In other embodiments multiplesecondary controllers may be used to control a single function.

In addition to systems required for sample, consumable, and fluidhandling the system may operate one or more computer apparatuses tofacilitate the functions described herein. Any of the elements in FIG.21 may use any suitable number of subsystems to facilitate the functionsdescribed herein. The subsystems shown in FIG. 20 are interconnected viaa system bus 775. Additional subsystems such as a printer 774, keyboard778, fixed disk 779 (or other memory comprising computer readablemedia), monitor 776, which is coupled to display adapter 782, and othersare shown. Peripherals and input/output (I/O) devices, which couple toI/O controller 771, can be connected to the computer system by anynumber of means known in the art, such as serial port 777. For example,serial port 777 or external interface 781 can be used to connect thecomputer apparatus to a wide area network such as the Internet, a mouseinput device, or a scanner. The interconnection via system bus allowsthe central processor 773 to communicate with each subsystem and tocontrol the execution of instructions from system memory 772 or thefixed disk 779, as well as the exchange of information betweensubsystems. The system memory 772 and/or the fixed disk 779 may embody acomputer readable medium.

The previous description provides exemplary embodiments only, and is notintended to limit the scope, applicability, or configuration of thedisclosure. Rather, the previous description of the exemplaryembodiments will provide those skilled in the art with an enablingdescription for implementing one or more exemplary embodiments. It isunderstood that various changes may be made in the function andarrangement of elements without departing from the spirit and scope ofthe invention. Several embodiments were described herein, and whilevarious features are ascribed to different embodiments, it should beappreciated that the features described with respect to one embodimentmay be incorporated within other embodiments as well. By the same token,however, no single feature or features of any described embodimentshould be considered essential to every embodiment of the invention, asother embodiments of the invention may omit such features.

Specific details are given in the previous description to provide athorough understanding of the embodiments. However, it will beunderstood by one of ordinary skill in the art that the embodiments maybe practiced without these specific details. For example, circuits,systems, networks, processes, and other elements in the invention may beshown as components in block diagram form in order not to obscure theembodiments in unnecessary detail. In other instances, well-knowncircuits, processes, algorithms, structures, and techniques may be shownwithout unnecessary detail in order to avoid obscuring the embodiments.

Also, it is noted that individual embodiments may be described as aprocess which is depicted as a flowchart, a flow diagram, a data flowdiagram, a structure diagram, or a block diagram. Although a flowchartmay describe the operations as a sequential process, many of theoperations can be performed in parallel or concurrently. In addition,the order of the operations may be re-arranged. A process may beterminated when its operations are completed, but could have alsoincluded additional steps or operations not discussed or included in afigure. Furthermore, not all operations in any particularly describedprocess may occur in all embodiments. A process may correspond to amethod, a function, a procedure, a subroutine, a subprogram, etc. When aprocess corresponds to a function, its termination corresponds to areturn of the function to the calling function or the main function.

Furthermore, embodiments may be implemented, at least in part, eithermanually or automatically. Manual or automatic implementations may beexecuted, or at least assisted, through the use of machines, hardware,software, firmware, middleware, microcode, hardware descriptionlanguages, or any combination thereof. When implemented in software,firmware, middleware or microcode, the program code or code segments toperform the necessary tasks may be stored in a machine readable medium.A processor(s) may perform the necessary tasks.

While detailed descriptions of one or more embodiments have been giveabove, various alternatives, modifications, and equivalents will beapparent to those skilled in the art without varying from the spirit ofthe invention. Moreover, except where clearly inappropriate or otherwiseexpressly noted, it should be assumed that the features, devices, and/orcomponents of different embodiments may be substituted and/or combined.Thus, the above description should not be taken as limiting the scope ofthe invention. Lastly, one or more elements of one or more embodimentsmay be combined with one or more elements of one or more otherembodiments without departing from the scope if the invention. Forexample, any suitable elements of an assay cartridge can be combinedwith any suitable elements of the various processing lanes in anysuitable manner, without departing from the spirit and scope of theinvention.

1. A computer readable medium, comprising code, executable by aprocessor, for implementing a method for operating a thermal cyclermodule, the method comprising: obtaining a predetermined temperature vs.time profile associated with a selected thermal cycler module in anarray of thermal cycler modules, the array of thermal cycler modulescomprising the selected thermal cycler module and a set of thermalcycler modules; and controlling, by a processor, the thermal cyclermodules in the set of thermal cycler modules so that their performancematches the predetermined temperature vs. time profile, each of thethermal cycler modules in the set of thermal cycler modules beingcontrolled using a source of variation between the thermal cyclermodules in the array.
 2. The computer readable medium of claim 1 whereinthe source of variation comprises an ambient temperature of the thermalcycler module, or a physical characteristic of the thermal cyclermodule.
 3. The computer readable medium of claim 1 wherein controllingthe thermal cycler modules comprises, for each module, adjusting h_(a),which is a thin film heater output at ambient temperature (° C./second)and k, which is a rate of heat transfer, using the following equation:dB/dt=h _(a) +k(Ta−B(t)), where dB/dt=change in block temp in degreesper second, Ta=ambient temperature (° C.), h_(a)=thin film heater outputat ambient temperature (° C./second), k=rate of heat transfer, andB(t)=the temperature of the thermal block at a given time t, whereindB/dt
 4. The computer readable medium of claim 3 whereinB(t)=(B(0)−(h_(a)/k)−Ta)e^(kt)+(ha/k)+Ta, where B(0)=starting blocktemperature at time=0.
 5. The computer readable medium of claim 3wherein k is adjusted using a blower.
 6. A computer readable medium,comprising code, executable by a processor, for implementing a method ofdriving a first thermal cycler in a predetermined thermal profile(B(t)), the first thermal cycler including a thermal block, a heaterthermally coupled to the thermal block, and a blower to direct air tothe thermal block, the method comprising: determining the rate of changeof the thermal block temperature with respect to time (dB/dt) as afunction of heater output (h_(a)), of blower heat transfer (k), and ofambient temperature (Ta); measuring the thermal block temperature;measuring the ambient temperature at the thermal cycler; and adjustingone of the heater output and the blower heat transfer according to amodeled relationship of:dB/dt=h _(a) +k(Ta−B(t)).
 7. The computer readable medium of claim 6wherein the step of adjusting one of the heater output and the blowerheat transfer includes adjusting the heater output and the blower heattransfer.
 8. The computer readable medium of claim 7 wherein the firstthermal cycler further includes a processor having a memory, theprocessor sensing the thermal block temperature and the ambienttemperature and controlling the heater output and the blower heattransfer, and the memory including a representation of the predeterminedthermal profile and a representation of the modeled relationship.
 9. Thecomputer readable medium of claim 8 wherein the processor controls theblower heat transfer by adjusting the power supplied to the blower. 10.The computer readable medium of claim 9 wherein the same predeterminedthermal profile is determined through evaluation of the rate of changeof the thermal block temperature with respect to time as a function ofheater output, of blower heat transfer, and of ambient temperature of aplurality of thermal cyclers and selection of a thermal profileachievable by each of the plurality of thermal cyclers as thepredetermined profile.
 11. The computer readable medium of claim 10wherein the response of the first thermal cycler matches the response ofa second thermal cycler.
 12. The computer readable medium of claim 11wherein first thermal cycler is disposed adjacent the second thermalcycler.